U.S. patent application number 10/656897 was filed with the patent office on 2004-06-17 for rapid assays for neurotransmitter transporters.
This patent application is currently assigned to Vanderbilt University. Invention is credited to Blakely, Randy D., DeFelice, Louis, Schwartz, Joel W..
Application Number | 20040115703 10/656897 |
Document ID | / |
Family ID | 32511251 |
Filed Date | 2004-06-17 |
United States Patent
Application |
20040115703 |
Kind Code |
A1 |
Schwartz, Joel W. ; et
al. |
June 17, 2004 |
Rapid assays for neurotransmitter transporters
Abstract
The invention describes the finding that
4-(4-dimethylaminostyrl)-N-methyl- pyridinium or ASP.sup.+ is a
fluorescent substrate that is transported by several
neurotransmitter transporters. Provided are methods for the
analysis of neurotransmitter transport and binding using ASP.sup.+.
The invention also provides rapid methods for screening for
modulators of neurotransmitter transport. As neurotransmitter
transporter defects are associated with numerous neurological
disorders, the invention also provides methods for treating
neurotransmitter transport-associated defects/conditions using the
modulators identified by the screening methods of the
invention.
Inventors: |
Schwartz, Joel W.;
(Nashville, TN) ; Blakely, Randy D.; (Brentwood,
TN) ; DeFelice, Louis; (Nashville, TN) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI L.L.P.
600 CONGRESS AVE.
SUITE 2400
AUSTIN
TX
78701
US
|
Assignee: |
Vanderbilt University
|
Family ID: |
32511251 |
Appl. No.: |
10/656897 |
Filed: |
September 5, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60408839 |
Sep 6, 2002 |
|
|
|
Current U.S.
Class: |
435/6.14 ;
435/7.2 |
Current CPC
Class: |
G01N 33/9406
20130101 |
Class at
Publication: |
435/006 ;
435/007.2 |
International
Class: |
C12Q 001/68; G01N
033/53; G01N 033/567 |
Goverment Interests
[0002] The government owns rights in the present invention pursuant
to grant numbers NS-34075 and NS-33373 from the National Institutes
of Health.
Claims
What is claimed is:
1. A method for measuring neurotransmitter transport activity in a
cell or cellular extract comprising: a) providing a cell that
expresses a neurotransmitter transporter or a cellular extract that
comprises a neurotransmitter transporter; b) exposing the cell or
the extract to ASP.sup.+; and c) measuring the transport of
ASP.sup.+; thereby measuring the activity of the neurotransmitter
transporter in the cell.
2. The method of claim 1, wherein measuring transport further
comprises measuring the kinetics of the neurotransmitter
transporter.
3. The method of claim 1, wherein measuring transport is in real
time.
4. The method of claim 1, wherein measuring the transport of
ASP.sup.+ is by fluorescence microscopy or using a fluorescent
plate reader.
5. The method of claim 1, wherein the time resolution of measuring
transport is 1 hour to 50 milliseconds.
6. The method of claim 1, wherein the cell is a neuronal cell.
7. The method of claim 1, wherein the cell is a blood platelet.
8. The method of claim 1, wherein the cell is a placental cell.
9. The method of claim 1, wherein the cell is a trophoblast.
10. The method of claim 1, wherein the neurotransmitter transporter
is an endogenously expressed transporter.
11. The method of claim 1, wherein the neurotransmitter transporter
is an exogenously expressed transporter.
12. The method of claim 1, wherein the neurotransmitter transporter
is a monoamine neurotransmitter transporter.
13. The method of claim 12, wherein the monoamine neurotransmitter
transporter is a norepinephrine transporter.
14. The method of claim 12, wherein the monoamine neurotransmitter
transporter is an epinephrine transporter.
15. The method of claim 12, wherein the monoamine neurotransmitter
transporter is a dopamine transporter.
16. The method of claim 12, wherein the monoamine neurotransmitter
transporter is a serotonin transporter.
17. A method of screening for agents that can modulate the activity
of a neurotransmitter transporter comprising: a) providing a cell
or cell extract that expresses a neurotransmitter transporter; b)
exposing said cell or cell extract to an agent that is a candidate
neurotransmitter transporter modulator; c) exposing the cell or
cell extract to ASP.sup.+; d) measuring the transport of ASP.sup.+;
and e) comparing the transport of ASP.sup.+ in said cell to the
transport of ASP.sup.+ in a cell or cell extract that has not been
exposed to the agent; thereby determining if the agent is a
modulator of neurotransmitter transporter activity.
18. The method of claim 17, further comprising the use of a
fluorescent plate reader to provide high-throughput screening of
agents.
19. The method of claim 17, wherein the neurotransmitter
transporter is a norepinephrine transporter, an epinephrine
transporter, a dopamine transporter or a serotonin transporter.
20. The method of claim 17, wherein said method is an in vivo
method.
21. The method of claim 17, wherein said method is an in vitro
method.
22. The method of claim 17, wherein measuring the transport of
ASP.sup.+ further comprises adding a quencher and measuring the
polarization of light in the presence and absence of the agent.
23. A method for the treatment of a nervous system disorder
comprising administering to a patient in need thereof a
neurotransmitter transporter modulator identified by the method of
claim 17.
24. The method of claim 23, wherein the nervous system disorder is
depression, hypertension, drug abuse, attention deficit disorder.
Description
[0001] The present application claims priority to co-pending U.S.
Provisional Patent Application Serial No. 60/408,839 filed Sep. 6,
2002. The entire text of the above-referenced disclosure is
specifically incorporated herein by reference without
disclaimer.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to the fields of
neurobiology and neurophysiology. More particularly, it concerns
the development of methods for measuring the transport and binding
of neurotransmitter transporters using ASP.sup.+ and other
fluorescent substrates. The invention also provides screening
methods for identifying modulators of neurotransmitter
transport.
[0005] 2. Description of Related Art
[0006] Neurotransmitters mediate signal transduction in the nervous
system and modulate the processing of responses to a variety of
sensory and physiological stimuli. An important regulatory step in
neurotransmission is the inactivation of a neurotransmitter
following its release into the synaptic cleft. This is especially
true for the biogenic amine and amino acid neurotransmitters.
Inactivation of a neurotransmitter is typically mediated by uptake
of the released neurotransmitter by neurotransmitter transporters
that are located on the presynaptic neuron or in some cases on
adjacent glial cells. Thus, neurotransmitter transporters are
central to the processing of information in the nervous system and
are associated with numerous neurological disorders.
[0007] For example, the neurotransmitter norepinephrine (also
called noradrenalin) transduces signaling in the central nervous
system that modulates attention, mood, arousal, learning, and
memory (Aston-Jones et al., 1999; Coull et al., 1999; Skrebitsky
and Chepkova, 1998; Hatfield and McGaugh, 1999). Norepinephrine
(NE) transporters (NETs) attenuate neuronal signaling via rapid
neurotransmitter clearance (Ressler and Nemeroff, 1999; Iversen et
al., 1967; Axelrod and Kopin, 1969; Blakely et al., 1991).
Norepinephrine transport is implicated in the pathology of major
depression, post-traumatic stress disorder and attention deficit
disorder (Ressler and Nemeroff, 1999; Southwick et al., 1999; Dow
and Kline, 1997; Biederman and Spencer, 1999). Therapeutic agents
that inhibit NET can elevate the concentration of norepinephrine in
the brain and periphery (Axelrod and Kopin, 1969; Bonisch, 1984;
Ramamoorthy et al., 1993; Galli et al., 1995; Corey et al., 1994;
Fleckenstein et al., 1999). Noradrenergic signaling in the
peripheral nervous system influences blood pressure and heart rate
(Jones, 1991; Jacob et al., 1999; Hartzell, 1980), and NET
inhibitors, such as cocaine and antidepressants, induce cardiac
complications (Watanabe et al., 1981; Clarkson et al., 1993;
Glassman et al., 1985).
[0008] Similarly other neurotransmitters such as epinephrine (E),
dopamine (DA), serotonin (SE), and their respective transporters
such as epinephrine transporters (ET), dopamine transporters (DAT),
and the serotonin transporters (SERT), mediate diverse aspects of
neuronal signaling and are involved in the pathology of numerous
nervous system related disorders. Thus, neurotransmitter
transporters are the targets of various therapeutic agents used in
the treatment of neurological disorders including, depression,
epilepsy, schizophrenia, Parkinson's disease, attention deficit
disorders, eating and sleeping disorders as well as some
neurodegenerative disorders. In some instances, treatment of these
disorders is mediated by the use of pharmaceutical agents that are
antagonists of a neurotransmitter transporter. Antagonists block
uptake and prolong and/or enhance the action of the
neurotransmitter. In other instances, treatment is mediated by use
of pharmaceutical agents that are agonists of a neurotransmitter
transporter. Agonists enhance uptake and rapidly clear the
neurotransmitter, thereby terminating its actions. For example,
imipramine, a blocker of SE and NE uptake, is used as an
antidepressants; benztropine, an antagonist of dopamine uptake,
temporarily alleviates the symptoms of Parkinson's disease; and
blockers of .gamma.-amino butyric acid (GABA) uptake are used in
the treatment of epilepsy.
[0009] Despite the relevance of neurotransmitter transporters, the
art is hindered by very limited methods that are used in studying
neurotransmitter transporter functions such as kinetics, affinity,
temporal and spatial aspects of transport, voltage dependence and
other transport mechanics (Galli et al., 1995; Corey et al., 1994;
DeFelice & Galli, 1998; Prasad and Amara, 2001). Methods used
to study neurotransmitter transport typically involve the use of
radiometric substrates to measure neurotransmitter accumulation.
For example, .sup.3H-labeled neurotransmitters are typically used
to study transport of serotonin, epinephrine, norepinephrine,
dopamine and the amino-acid transmitters (see for example U.S. Pat.
No. 5,424,185; Bonisch 1984; Bonisch and Harder, 1986; Hadrich et
al., 1999). Although radiolabel techniques offer high specificity,
these approaches have significant limitations such as poor time and
spatial resolution. In addition, none of these methods have the
intrinsic capability to distinguish substrate binding from
transport in the same assay. For example, non-permeating
radiolabled molecules that bind neurotransporters can characterize
binding and count transporters, and permeating radiolabled
molecules can characterize transport, however, because of the poor
time resolution of radiometric assays, it is not possible to study
binding and transport during the same experiment. Furthermore,
these methods are not applicable for studying transport function in
single mammalian cells. Although electrophysiology and amperometry
alleviate some of these constraints, eletrophysiology although
rapid (in the millisecond time resolution) has poor substrate
selectivity, while amperometry has the reverse characteristics
(DeFelice and Galli, 1998; Galli et al., 1998).
[0010] Several other studies involved the use of fluorescent
analogs of neurotransmitters for the study of neurotransmitter
transporters. For example, Hadrich and colleagues generated
fluorescent NE and nisoxetine analogs to image neuroblastomas
(Hadrich et al., 1999), and Bruns (1998) used a autofluorescent
analog of serotonin (5-HT), 5,7-dihydrotryptamine to identify a
serotonin uptake current in leech neurons, however, these
fluorescent compounds were also unable to distinguish substrate
binding from transport. Thus, new methods for the analysis of
neurotransmitter transport finction are highly desirable.
[0011] In addition, the art also lacks cost effective and rapid
screening methods to identify modulators of neurotransmitter
transporters that may be useful as therapeutic agents in the
treatment of nervous system disorders.
SUMMARY OF THE INVENTION
[0012] The present invention overcomes the defects in the art and
provides methods for the analysis of neurotransmitter transporters
based on the use of fluorescent substrates. The invention also
provides screening methods to identify agents that can modulate
neurotransmitter transporters.
[0013] In particular embodiments, the present invention provides
that 4-(4-dimethylaminostyrl)-N-methylpyridinium (ASP.sup.+) is
transported by neurotransmitter transporters such as DAT, NET and
SERT and can used for measuring neurotransmitter transport.
[0014] Thus, the present invention provides methods for measuring
neurotransmitter transport in a cell or cellular extract comprising
providing a cell that expresses a neurotransmitter transporter or a
cellular extract that comprises a neurotransmitter transporter;
exposing the cell or the extract to ASP.sup.+; and measuring the
transport of ASP.sup.+; thereby measuring the transport of the
neurotransmitter in the cell.
[0015] The term "neurotransmitter transporter" or "transporter" is
used herein to describe a membrane protein which, under physiologic
conditions, is at least substantially specific for the transport of
at least one neurotransmitter.
[0016] The measurement of ASP.sup.+ transport is performed by
fluorescence microscopy. In other embodiments, one may measure
transport by using a fluorescent plate reader. In some embodiments
of the invention, measuring transport further comprises measuring
the kinetics of the neurotransmitter transporter. In yet other
embodiments, measuring transport is in real time. In some specific
embodiments, the time resolution of measuring transport is 1 hour
to 50 milliseconds. In yet other embodiments, one may measure the
voltage dependence, the turnover rate, the surface expression,
and/or binding constants of the transporter. In still other
embodiments, measuring transport includes characterizing the
kinetics, affinity, uptake of neurotransmitter, retention or
accumulation of neurotransmitter or other substrate, regulation by
phosphorylation or other biochemical modifications.
[0017] In some embodiments, measurement of transport
characteristics is achieved in cells transfected to express the
transporter. In other embodiments, the measurement of transport is
in non-transfected neurons in tissue culture and allows the
characterization of endogenous transport regulation and
function.
[0018] In some embodiments, transport is measured in a single cell.
In other embodiments, transport is measured in a single neuronal
process. In yet other embodiments, transport is measured in more
than one cell or in a population of cells. Transport may be
measured simultaneously from numerous samples using multi-well
formats, fluorescent plate readers, and other automated methods
known in the art.
[0019] The neurotransmitter transporter maybe endogenously
expressed by the cell. In alternative embodiments, the
neurotransmitter transporter is expressed exogenously by the cell.
Recombinant DNA technology may be used to express any
neurotransmitter transporter exogenously in a cell using methods of
molecular biology as are known to one of skill in the art. The
specification provides detailed description of methods used for
exogenous expression infra. One of skill in the art would be well
equipped to construct an expression vector that expresses nucleic
acids encoding any neurotransmitter transporter using standard
molecular biology techniques (see, for example, Maniatis et al.,
1988 and Ausubel et al., 1994, both incorporated herein by
reference). Furthermore, Galli et al. (1995), Ramamoorthy (1998),
and U.S. Pat. Nos. 5,312,734, 5,418,162, & 5,424,185, all
incorporated herein by reference, describe numerous nucleic acids,
constructs and host cells used to express neurotransmitter
transporters.
[0020] In specific embodiments, the neurotransmitter transporter is
a monoamine neurotransmitter transporter and may be a
norepinephrine transporter, an epinephrine transporter, a dopamine
transporter, or a serotonin transporter. It is also contemplated
that the methods of the present invention will be applicable to
other neurotransmitter transporters including GABA transporters,
glutamate transporters, and glycine transporters, provided the
fluorescent substrate that is used is transported by these
transporters.
[0021] In other specific embodiments, the cell is a neuronal cell.
In yet other specific embodiments, the cell expressing the
neurotransmitter transporter may be a blood platelet, a placental
cell or a trophoblast.
[0022] In embodiments where cellular extracts comprising one or
more neurotransmitter transporter are used, the cellular extracts
may further comprise cell membranes. The term "cellular extract" is
defined herein as a complex biochemical and aqueous solution
comprising one or more neurotransmitter transporter(s). It is
contemplated that the cellular extract may also comprise endogenous
regulators and modulators of neurotransmitters. Typically, the
neurotransmitter will be comprised in a cell membrane. Cells
expressing neurotransmitter transporters may be lysed to provide
cell membranes and/or cellular extracts. The cellular extract may
be from any cell that endogenously expresses a neurotransmitter
transporter, such as a neuronal cell, a blood platelet, a placental
cell, a trophoblast or may be from any cell that is engineered to
exogenously express a neurotransmitter transporter. In some
specific embodiments, the cell membranes may be from cells that are
engineered to exogenously express a single type of neurotransmitter
transporter and therefore is free of other neurotransmitter
transporters. This allows the analysis of only one type of
neurotransmitter transporter in isolation. One may also analyze the
effects of other molecules or interacting proteins on the
neurotransmitter transporter.
[0023] In other embodiments, it is contemplated that one may use
analogs of ASP.sup.+ such as,
4-(4-diethylaminostyrl)-N-methylpyridinium iodide
(4-Di-2-ASP.sup.+), 4-(4-dimethylaminostyrl)-N-methylpyridinium
iodide (4-Di-1-ASP.sup.+),
2-(4-dimethylaminostyrl)-N-methylpyridinium iodide (DASPMI),
2-(4-dimethylaminostyrl)-N-ethylpyridinium iodide (DASPEI), and
other members of the styryl pyridinium family of dyes (Herrera and
Banner, 1990). It is also contemplated that one may also use dyes
such as 3, 3'diethyloxadicarbocyanine iodide (DIOC) or acridine
orange-10-nonyl bromide (NAO) (Herrera and Banner, 1990).
[0024] The invention also provides methods of screening for agents
that can modulate the activity of a neurotransmitter transporter
comprising providing a cell or cellular extract that expresses a
neurotransmitter transporter; exposing the cell or cellular extract
to an agent that is a candidate neurotransmitter transporter
modulator; exposing the cell or cellular extract to ASP.sup.+;
measuring the transport of ASP.sup.+; and comparing the transport
of ASP.sup.+ in the cell or cellular extract to the transport of
ASP.sup.+ in a cell or cellular extract that has not been exposed
to the agent, thereby determining if the agent is a modulator of
activity of the neurotransmitter transporter.
[0025] It is contemplated that the screening methods will be
automated to provide high-throughput screening of agents. For
example, in some embodiments, the methods comprise the simultaneous
screening of multiple agents with potential neurotransmitter
transporter modulatory activities. This may be achieved by addition
of reagents/components of the assay using robotic fluid delivery
(see Example 3 and FIG. 9, FIG. 10 and FIG. 11 for the use of
FLEXstation (Molecular Devices)); the analysis of multiple samples
in multi-well formats; using a fluorescent plate reader (also see
Example 3 for examples of multiwell assays, plate readers and
computerized software for data analysis) as well as other
automation methods known in the art. Other examples of methods of
automated equipment and assay procedures for membrane associated
proteins such as ion channels are described in U.S. Pat. Nos.
6,127,133 and 5,670,113, the contents of which are incorporated by
reference herein.
[0026] The screening methods of the invention may be in vitro or in
cyto screening methods that use cells, cell lines, recombinant
cells or cellular extracts thereof. Any cells which express a
neurotransmitter transporter may be used in the screening methods,
either as whole cells or lysed to provide cell membranes or cell
extracts as described above.
[0027] The screening methods may also be performed using in vivo
methods, for example using animal models. In some cases, the
animals may be transgenic animals. It is contemplated that one may
use animals such as mice or C. elegans as the genetics of these
systems as well as methods for establishing transgenics are well
known in these animals. Animals expressing certain types of
neurotransmitter transporters can be provided with candidate
modulatory agents and the transport of ASP.sup.+ or other
fluorescent substrate can be imaged in vivo or in situ. General
methods for in vivo imaging using ASP.sup.+ are described in
Herrera and Banner (1990), and in Herrera et al. (1990), the
contents of both are incorporated herein by reference. In situ
methods for analysis of ASP.sup.+ are exemplified by the work by
Ullrich and colleagues (Pietruck and Ullrich, 1995; Rohlicek and
Ullrich, 1994; the contents of both are incorporated herein by
reference). These methods may be suitably modified with the other
teachings of the specification. The present invention contemplates
the use of these methods in conjunction with the screening methods
described herein.
[0028] In some embodiments, measuring the transport of ASP.sup.+
further comprises adding a quencher and measuring the polarization
of light in the presence and absence of the modulatory agent.
Quenchers allow reduction of unwanted background signals from the
solution and also test the depth of ASP penetration in the
membrane-bound transporter. Polarized light measures the mobility
of the ASP molecules in solution as well as ASP molecules bound to
the transporter, thus reporting the extent and location of
binding.
[0029] Thus, the methods of the invention provide rapid assays for
screening agents and are expected to provide therapeutic agents
that are potentially useful for the treatment of disorders
associated with neurotransmitter transporter function. By measuring
and comparing changes in transport of ASP.sup.+ (or other
fluorescent substrate), in the presence and absence of the
investigatory or candidate agent, one can further evaluate the role
of the compound. For example, where an agent causes a change in a
specific finction or transport mechanism, the agents may be further
screened to determine the specific extent to which the compound
acts as either an agonist or antagonist. It is also contemplated
that the modulators may not be antagonists or agonists per say and
may mediate their function by other mechanisms.
[0030] Agents that may be screened include substances or compounds
that are naturally occurring such as, plant products or extracts
and animal derived products, including macromolecular entities such
as polypeptides, polynucleotides, lipids, sugars, small entities
such as ligands, neurotransmitters, amino acids, elemental
compounds as well as other organic compounds and inorganic
compounds. The candidate agents may also be man-made substances
such as synthetic compounds or pharmaceutical formulations of
natural or synthetic agents. The synthetic compounds and/or natural
products also include substances that are either part of a crude
mixture or are partially to completely purified/isolated.
[0031] The invention also provides methods for the treatment of a
nervous system disorder comprising administering to a patient in
need thereof a neurotransmitter transporter modulator identified by
the screening methods described above. These methods are
contemplated to be useful in treatment of nervous system disorders
such as, depression, hypertension, drug abuse and addiction,
attention deficit disorder, neurodegeneration and others. It is
envisioned that the therapeutic agents identified herein may be
administered with other standard therapies that are normally used
to treat the disorder or condition. In some embodiments of this
facet, the modulatory agent maybe administered simultaneously,
prior to or after the administration of the other drug or therapy
that is routinely used to treat the disorder.
[0032] As used herein the specification, "a" or "an" may mean one
or more. As used herein in the claim(s), when used in conjunction
with the word "comprising", the words "a" or "an" may mean one or
more than one. As used herein "another" may mean at least a second
or more.
[0033] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] The following drawings form part of the present
specification and are included to firther demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0035] FIG. 1. MPP.sup.+ and ASP.sup.+ inhibit NE accumulation.
[.sup.3H]NE accumulation was measured in the hNET-transfected
HEK-293 cells in the presence of increasing concentrations of
ASP.sup.+ or MPP.sup.+ and normalized to similar data in the
absence of an inhibitor. Non-specific activity was determined by
application of 10 .mu.M desipramine. The data were fit to NE
transport remaining by the equation
y=100/(1+([I]/IC.sub.50).sup.n), where [I] is the concentration of
ASP.sup.+ or MPP.sup.+ and K.sub.i values were determined using
Cheng-Prusoff correction for substrate concentration. The fits
yield K.sub.i (ASP.sup.+)=780.+-.77 nM and Ki (MPP.sup.+)=600.+-.67
nM. Values are represented as means.+-.S.E.M., N=5.
[0036] FIGS. 2A-2C. Cells expressing NET accumulate ASP.sup.+.
Single cells are visualized by DIC microscopy (top row) and
accumulation is measured by an increase in ASP.sup.+ fluorescence
under confocal microscopy (bottom row). Images were taken at 0, 3
and 180 seconds after exposure to 800 nM ASP.sup.+ (FIGS. 2A, 2B
& 2C). In the upper panels, fluorescence images were projected
onto the DIC image of the corresponding cells to identify cells
surface (S) and interior (I). The color gradient represented in
FIG. 2A denotes the ASP.sup.+ intensity values (red being most
intense).
[0037] FIGS. 3A-3L. Monoamine transporters interact with ASP.sup.+.
Like HEK-hNET cells hDAT and hSERT transfected cells accumulate
ASP.sup.+. hNET (FIGS. 3A, 3B & 3C), hDAT (FIGS. 3D, 3E &
3F), hSERT (FIGS. 3G, 3H & 3I) and HEK-293 (FIGS. 3J, 3K &
3L) were exposed to 2 mM ASP for the times indicated in FIGS. 3J,
3K & 3L. The color gradient represented in FIG. 3J denotes the
corresponding ASP.sup.+ intensity values.
[0038] FIG. 4. ASP.sup.+ photo-bleaching. Transfected HEK-HNET and
parental HEK-293 cells were exposed to 2 mM ASP.sup.+ for 180 sec
followed by a wash. Images were taken from at least 40 individual
cells per dish (cells defined by the corresponding DIC image) from
4 separate dishes. The average pixel intensity.+-.S.E.M over all
cells is plotted on the y-axis in arbitrary fluorescence units
(AFUs). After ASP.sup.+ was removed, photo-bleaching was assessed
at 0.3 Hz and 12 Hz for hNET-293 cells and 12 Hz for HEK-293 cells.
Similar data were obtained up to 20 Hz and normalized to the
fluorescence intensity maximums before and after ASP.sup.+
removal.
[0039] FIGS. 5A-5D. Phase I represents ASP.sup.+ binding; phase II
represents ASP.sup.+ transport. Line scans across the center of
HEK-hNET (FIG. 5A) and HEK-293 (FIG. 5B) cells were taken at 0, 3
and 180 seconds after exposure to 2 mM ASP.sup.+. Phase I, which
refers to the initial increase in fluorescence as identified in DIC
images, is localized to the cell surface (arrows), and the slower
phase II registers with the cytosol (between arrows). In FIG. 5C,
hNET-HEK cells are exposed to 2 mM ASP.sup.+ under blue polarized
light and red images were collected at 0.degree. and 90.degree.
with respect to the incident polarization. Polarized images from
ASP.sup.+ in solution, in the cytosol, and at the cell surface were
collected from at least 40 cells in 4 dishes in FIG. 5D.
[0040] FIGS. 6A-6D. Desipramine displaces phase I ASP.sup.+ and
arrests phase II transport. The top panels show confocal images of
confluent HEK-hNET cells exposed to 2 mM ASP for 60 sec (FIG. 6A)
followed by application of 10 mM desipramine (FIG. 6B). From
similar data, FIG. 6C shows three separate HEK-hNET cells exposed
to 2 mM ASP.sup.+ for 60, 120 or 180 seconds followed by rapid
application of 10 mM desipramine. Data integrated over the entire
cell were collected at 0.3 Hz to avoid photo-bleaching. The top
line represents the slope of phase II and the bottom line the
corresponding increase in sequestered ASP.sup.+ at each time (only
three of six are shown). FIG. 6D shows the temperature dependence
of phase I and phase II slopes in 2 mm ASP.sup.+. The slopes of
phase I and II are normalized to room temperature. The color
gradient in FIG. 6B represents ASP.sup.+ intensity.
[0041] FIGS. 7A-7C. ASP.sup.+ pharmacology. The bars in FIG. 7A
correspond to normalized slopes of phase I and II under various
treatments. Na.sup.+ and Cl.sup.- are replaced with NMDG+ and
acetate, respectively. FIG. 7B shows the average pixel intensity
from whole-cell confocal images as described in FIG. 3. External
ASP.sup.+ is varied as indicated. FIG. 7C plots the kinetics of
phase I and II as a fuiction of ASP.sup.+ concentration. Values are
represented at normalized slopes.+-.STD of four experiments with
100 cells per experiment.
[0042] FIGS. 8A-8C. ASP.sup.+ accumulation in superior cervical
ganglia (SCG) cells. FIG. 8A quantifies desipramine-sensitive
accumulation by monitoring the increase in intracellular
fluorescence within individual neurons. Comparing FIG. 8B (without
desipramine (DS) and FIG. 8C (with DS) shows that ASP.sup.+
accumulation in superior cervical ganglia (SCG) cells is
desipramine sensitive. The color gradient represented in FIG. 8A
denotes the color range corresponding to the intensity values.
[0043] FIG. 9. Evidence of accumulation of ASP.sup.+ by monoamine
transporters in transfected HEK-293 cells. Cells were plated in 96
well multiwell dishes and exposed to ASP.sup.+ in the presence of
the external quencher Trypan Blue. ASP.sup.+ addition was performed
using the. robotic fluid delivery abilities of the FLEXstation
(Molecular Devices) and the amount of ASP.sup.+ accumulation
determined at 15 minutes on the Flexstation. An equal number of
parental and transfected cells were compared in parallel.
[0044] FIGS. 10A-10C. Concentration-response profiles for ASP.sup.+
accumulation in monoamine transporter transfected HEK-293 cells.
Increasing concentrations of ASP.sup.+ were delivered to plated,
adherent cells and the accumulation of ASP.sup.+ inside cells
quantitated on the FLEXstation as described in Example 3. NET (FIG.
10A), DAT (FIG. 10B), and SERT (FIG. 10C), demonstrate
concentration-dependent accumulation of ASP.sup.+ with accumulation
well fit as a one site, nonsaturable transport process.
[0045] FIGS. 11A-11B. ASP.sup.+ accumulation in transfected cells
is blocked by monoamine transporter antagonists. Transport assays
with ASP.sup.+ on the FLEXstation were performed either with or
without competing antagonists. Desipramine, a NET-specific
antagonist, blocked accumulation of ASP.sup.+ by NET (FIG. 11A),
whereas GBR-12935, a specific DAT antagonist blocked ASP.sup.+
accumulation by DAT (FIG. 11B).
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0046] Although neurotransmitter transporters are central to
neuronal signal processing and have been implicated in various
nervous system related disorders, the art lacks methods that
effectively characterize neurotransmitter function and
activity.
[0047] The present inventors have found that the fluorescent
substrate 4-(4-dimethylaminostyrl)-N-methylpyridinium (ASP.sup.+)
is transported by NET, DAT and SERT. The present inventors have
developed methods to measure neurotransmitter transport mechanisms
using ASP.sup.+ and fluorescence microscopy. The present inventors
have shown that the ASP.sup.+ fluorescence assays provide
mechanistic information about transport including the kinetics of
the Na.sup.+- and Cl.sup.--dependent transport and the kinetics
involved in blockade of transport by antagonists.
[0048] Furthermore, the inventors have developed rapid screening
methods that utilize fluorescent substrates such as ASP.sup.+ to
identify modulators of neurotransmitter transporters.
Pharmaceutical formulations of the modulators of neurotransmitter
transporters identified by the methods of the present invention may
be used to treat various nervous system disorders that are caused
by defects in neurotransmitter uptake and retention mechanisms.
Therefore, the invention also provides methods of treatment of
nervous system disorders by using neurotransmitter transport
modulators that are identified by the screening methods of the
present invention.
A. ASP.sup.+
[0049] ASP.sup.+ [4-(4-(dimethylamino)styrl)-N-methyl-pyridinium]
is a permanent, positively-charged fluorescent dye originally used
for the vital staining of mitochondria and nerve terminals
(Morozova et al., 1981). ASP.sup.+ fluoresces visible light and is
a substrate for organic cation transporters (OCT) (Hohage etal.,
1998; Stachon et al., 1996). ASP.sup.+ is structurally related to
1-methyl-1,2,3,6-tetrahydopyridinium (MPP), which is a neurotoxic
metabolite of MPTP that induces dopamine transporter DAT)-dependent
neurotoxic degeneration of the substantial nigra (Gainetdinov et
al., 1997).
[0050] Using time-resolved fluorescence microscopy, the present
inventors have demonstrated ASP.sup.+ accumulation in human
embryonic kidney cells (HEK) expressing human norepinephrine (NE)
transporters (hNET). ASP.sup.+ accumulation has sub-.mu.M potency
for HNET, requires Na and Cl, is blocked by cocaine and
desipramine, and is competed for by NE. The inventors have measured
ASP.sup.+ accumulation from single hNET-transfected HEK cells with
a 50 msec time resolution. The present inventors have also shown
that ASP.sup.+ is also a substrate for the dopamine and serotonin
transporters. It is contemplated that ASP.sup.+ will also be useful
in the study of epinephrine transport. ASP.sup.+ fluorescent
microscopy permits localization of transport activity in single
cells and neuronal processes. The ASP.sup.+ fluorescent microscopy
methods of the invention also permits analysis of many cells, while
retaining information about single cells. The ASP.sup.+
fluorescence assays of the invention provide detailed mechanistic
information about transport. For example, temporal and spatial
resolution of transport, transport kinetics, affinity for
substrate, turnover rates, surface expression, and binding
constants may be measured. Furthermore, features such as voltage
dependence of neurotransmitter accumulation can be assessed under
voltage clamp using ASP.sup.+ fluorescent microscopy.
B. Neurotransmitter Transporters
[0051] As described earlier, neurotransmitter transporters are
responsible for the uptake of neurotransmitters from the synaptic
cleft and thereby are responsible for the regulation of
neurotransmission. Transporter proteins in the plasma membranes of
neurons and glia also participate in vital nutrient and osmolyte
acquisition. Neurotransmitter transporters are typically ion
dependent, have high-affinity/specificity for one neurotransmitter,
and are temperature and pH sensitive.
[0052] Chemical signaling by small molecule neurotransmitters,
including DA, NE, E, SE (or 5HT), glutamate, glycine, and GABA, is
terminated by transporter-mediated clearance (Rudnick and Clark,
1993). Disruption of transporter function, mediated by genetic
mutations, pathological conditions or drugs of abuse, can elevate
or rapidly decrease extracellular neurotransmitter levels, perturb
presynaptic transmitter homeostasis, and trigger significant
alterations in physiology and behavior (Giros et al., 1996; Pelham,
1997). For example, psychoactive agents such as cocaine and the
amphetamines compete with the neurotransmitter substrates of the
DA, NE, and SE transporters, and their addictive potential has been
attributed to DAT blockade (Kuhar et al., 1991). In contrast, NET
and SERT antagonists such as imipramine, desipramine, fluoxetine,
and sertraline are important agents in the treatment of mood
disorders, particularly depression (Barker and Blakely, 1996).
Cloning and molecular analysis of neurotransmitter transporters has
also shown that genetic mutations and variations are associated
with some neuronal disorders and some forms of addictions to
substances of abuse.
[0053] However, the study of neurotransmitter transporters is
severely limited by methods that utilize radiolabeled
neurotransmitters or fluorescent analogs of neurotransmitters, all
of which have so far been incapable of distinguishing substrate
binding from transport. The methods of the present invention
provide better understanding of transport with a superior time and
spatial resolution and at the level of a single cell, if required.
Thus, the present methods provide better characterization of
transport mechanics of transporters. This is also relevant in the
case of diseases associated with mutations of neurotransmitter
transporters as the methods will aid in better understanding the
physiological basis of neuronal disorders caused by mutant
transport molecules in comparison to normal molecules. The methods
of the invention are also important with regard to providing a
better understanding of transport changes caused by numerous
addictive agents and therapeutic agents that target
neurotransmitter transporters.
[0054] In addition, the screening methods of the invention, provide
rapid screening and identification of novel modulators of
neurotransmitter transport. Such assays are also beneficial for
screening for modulators of mutant transporters that are expressed
in patients with genetic neuronal disorders. It is contemplated
that such methods will be useful in identifying therapeutic agents
specifically tailored to treat an individual patient. As
neurotransmitter transporters are also associated with addiction to
drugs of abuse and alcohol the screening methods of the invention
are contemplated to provide therapeutic agents that will be
effective in reversing such addictions.
[0055] i. Norepinephrine Transporters (NET)
[0056] NET is a member of a large family of Na.sup.+ and Cl.sup.-
dependent transporters (Blakely et al., 1991; Masson et al., 1999),
exhibits a sub-micromolar substrate potency and can concentrate NE
against its concentration gradient. NET accumulates NE by coupling
the substrate and co-transported ions at a proposed stoichiometry
of 1NE/1Na.sup.2+/1Cl.sup.- (Ressler and Nemeroff, 1999;
Ramamoorthy et al., 1993; Bonisch and Harder, 1986).
[0057] Approximately 70-90% of the NE released into synapses is
estimated to be cleared using NET. NE uptake by NET is
competitively inhibited by various drugs of abuse such as
amphetamine and cocaine, and antidepressants (e.g., desipramine,
imipramine, venlafaxine, mirtazapine, reboxetine, bupropion),
thereby resulting in an elevation of the synaptic concentrations of
NE which results in potentiation of the activation of postsynaptic
receptors. Other evidence has shown that treatments with drugs that
alter noradrenergic transmission can cause an up- or downregulation
of NET, which in turn causes changes in the sensitivity to
endogenous catecholamines.
[0058] NET was isolated by expression cloning in 1991, and the gene
was found to be located on human chromosome 16q 12.2 (Pacholczyk et
al., 1991). The NET gene is encoded by 14 exons, which span 45 kb
from the start to the stop codon (Porzgen et al., 1996). The
nucleotide and deduced amino acid sequence of the transporter
predict a protein of 617 amino acids, containing 12
membrane-spanning domains. The organization of the protein is
highly homologous to that of other neurotransmitter transporters
including those transporting dopamine, epinephrine, serotonin and
gamma-aminobutyric acid (GABA), which are members of a family of
sodium- and chloride-dependent transport proteins in the plasma
membranes of neurons and glial cells. Analysis of the NET gene and
protein has facilitated the investigation of its potential role in
psychiatric and other neuronal disorders. At least 13 genetic
variants of NET have been identified so far by methods such as
single-stranded conformational polymorphism analysis (Stober et
al., 1996; Samochowiec et al., 2001; Kitayama et al., 2001).
[0059] ii. ET
[0060] The neurotransmitter NE is converted to epinephrine (E or
Epi) in some neuronal cells, such as sympathetic neurons, and
released as the primary neurotransmitter. Blakely and colleagues
have cloned an E transporter (ET) cDNA from the bullfrog (Rana
catesbiana) paravertebral sympathetic ganglia and characterized its
functional properties via heterologous expression in non-neuronal
cells (Apparsundaram et al., 1997; Blakely and Apparsundaram,
1998). A 2514 bp cDNA corresponding to the frog ET (fET), was
identified and sequence analysis revealed an open reading frame
coding for a protein of 630 amino acids. The fET protein sequence
has a 75, 66, and 48% amino acid identity with human NET, DAT, and
SERT, respectively. Transfection of HeLa cells with fET confers
Na.sup.+- and Cl.sup.--dependent catecholamine uptake. Uptake of
[.sup.3H]NE by fET is inhibited by catecholamines in a
stereospecific manner and fET-mediated transport of catecholamines
was found to be sensitive to cocaine and other tricyclic
antidepressants. Although the human ET has not yet been cloned, the
methods of the present invention are envisioned to be effective to
study the transport characteristics of any human protein that
transports epinephrine.
[0061] iii. Dopamine Transporter (DAT)
[0062] The dopamine transporter (DAT) is a member of the subfamily
of monoamine transporters with numerous common topological
structures and significant amino acid sequence homology. DAT has
been identified as located on the distal end of chromosome 5
(5p15.3) (Giros et al., 1992). Kawarai et al. (1997), isolated and
characterized the human DAT gene (hDAT) including about 1 kb of
5'-flanking region. The hDAT gene spans over 64 kb, consisting of
15 exons separated by 14 introns. The intron-exon structure of the
hDAT gene is most similar to that of the human NET gene. Promoter
sequence analysis demonstrated a `TATA`-less, `CAT`-less and
G+C-rich structure. Two E box and several Sp-1-binding sites exist
in the promoter region. These structural features are similar to
that of the human D1A dopamine receptor gene and the human
monoamine oxidase A gene. The DAT gene encodes for a 620-amino acid
protein with a calculated molecular weight of 68,517 (Giros et al.,
1992) and is associated with numerous neuropsychiatric disorders
(Bannon, 2001). Examples of neurological diseases involving
dopamine transporter function include schizophrenia, addiction
disorders, attention deficit hyperactivity disorder (ADHD),
psychoses, Tourette's syndrome, or Parkinson's disease.
[0063] iv. SERT
[0064] The serotoninergic system modulates numerous behavioral and
physiological functions and has been associated with control of
mood, emotion, sleep and appetite. Synaptic serotonin (SE), also
called 5-hydroxytryptamine or 5HT, concentration is controlled by
the serotonin transporter (SERT) which is involved in reuptake of
serotonin into the pre-synaptic terminal. The cloning of the human
SERT protein by Ramamoorthy et al., (1993), shows that human SERT
is encoded by a single gene that is localized to chromosome
17q11.1-17q12 and encodes for a 630-amino acid protein. The hSERT
is a Na.sup.+- and Cl.sup.--coupled serotonin transporter and has
been found to be expressed on human neuronal, platelet, placental,
and pulmonary membranes (Ramamoorthy et al., 1993).
[0065] The SERT has been associated with depression and anxiety
(Soubrie, 1988; Barnes, 1988); obesity (Blundell, 1986; Silverstone
et al., 1986); alcoholism (Gill et al., 1987; Naranjo et al.,
1987); postanoxic intention myoclonus (Van Woert et al., 1976);
acute and chronic pain (Le Bars, 1988); as well as sleep disorders
(Koella, 1988). SERT has also been shown to mediate behavioral
and/or toxic effects of cocaine and amphetamines (Ramamoorthy et
al., 1993). A variety of specific serotonin reuptake inhibitors
(SSRIs) such as fluoxetine and paroxetine have been developed for
the treatment of depression (reviewed in Scholss, 1998). However,
as Schloss points out, the art lacks a detailed understanding of
the mode of action of these antidepressant drugs on their target,
the SERT protein. Furthermore, although several drugs that target
the SERT have been identified the art still lacks effective drugs
for the treatment and alleviation of depression and other
neurological disorders.
[0066] Recent research has shown that polymorphisms in the
promoters of SERT's are a risk factor for susceptibility to
depression (Neumeister et al., 2002). Other studies have also shown
the association of variants of SERT's to other disorders. For
example, association for allele 12 of the variable number tandem
repeat (VNTR) in the second intron of the SERT gene and
schizophrenic disorders has been shown (Tsai et al., 2002).
C. Methods of Measurement of Transport
[0067] The present invention provides methods for the measurement
of transport of neurotransmitter transporters including the
transporters for biogenic amines such as serotonin, dopamine,
epinephrine, norepinephrine. It is contemplated that these methods
are also applicable to transporters of the amino acids
neurotransmitters such as L-glycine and L-glutamate, L-aspartate,
and g-aminobutyric acid (GABA). In some embodiments, the present
invention provides a novel and rapid method for analysis of
transport by a neurotransmitter transporter that comprises the
measurement of uptake and/or accumulation of ASP.sup.+ that is
specifically taken up by the transporter. The methods of
measurement involve fluorescence microscopy. In other embodiments,
other fluorescent substrates may be used, some of which are
contemplated to be analogs of ASP.sup.+ and others are contemplated
to be analogs of other native neurotransmitters.
[0068] i. Microscopy
[0069] Fluorescent microscopy is used to measure transport using
ASP.sup.+ which is a fluorescent substrate for several
neurotransmitter transporters. Cells that either endogenously or
exogenously express a neurotransmitter are isolated and plated on
glass bottom Petri-dishes or multi-well plates that may typically
be coated with poly-L-lysine or any other cell adhesive agent.
Cells are typically cultured for three or more days. The culture
medium is then aspirated and the cells are mounted on a Zeiss 410
confocal microscope. During the confocal measurement cells remain
without buffer for approximately thirty seconds. Background
autofluorescence is established by collecting images for ten
seconds prior to the addition of the buffer and ASP.sup.+. As
ASP.sup.+ has a large Stoke shift between excitation (1.sub.max=488
nm) and emission maxima (1.sub.max=610 nm), the argon laser is
tuned to 488 nm and the emitted light filtered with a 580-630 nm
band pass filter (1.sub.max=610nm). The substantial red shift can
be exploited to reduce background auto-fluorescence produced in the
absence of substrate. The gain (contrast) and offset (brightness)
for the photomultiplier tube (PMT) may be set to avoid detector
saturation at the higher ASP.sup.+ concentrations that may be used
in certain experiments. The effects of photo-bleaching on ASP.sup.+
accumulation may also be determined by examining the rate of
ASP.sup.+ accumulation and decay at various acquisition rates. In a
constant pool of ASP.sup.+, rates as high as 20 Hz (50 msec/image)
can be set.
[0070] ii. Fluorescence Anisotropy Measurements
[0071] To evaluate ASP.sup.+ binding to the surface membranes,
cells expressing a neurotransmitter transporter may be exposed to
ASP with horizontal polarizer (see, for example, as in FIG. 5C),
with the polarizer rapidly switching to the vertical position.
Cells may be imaged with alternating polarizations for 3 minutes to
measure light intensity in the horizontal (I.sub.h) and vertical
(I.sub.v) positions in order to calculate the anisotropy ratio,
r=(I.sub.v-gI.sub.h)/(I.sub.v+2 g I.sub.h). The factor g may be
determined by using a half wave plate as described by Blackman et
al. (1996). In this formulation, r=0.4 implies an immobile light
source. Surface anisotropy can be measured at the cell
circumference over 1 pixel width (0.625 mm). Cytosolic anisotropy
can be measured near the center of the cell, approximately 5 pixel
widths from the membrane.
[0072] iii. Image Analysis
[0073] The fluorescent images may be processed using suitable
software. For example, fluorescent images were processed using
MetaMorph imaging software (Universal Imaging Corporation,
Downington PA). Fluorescent accumulation was established by
measuring the average pixel intensity of time resolved fluorescent
images within a specified region identified by the DIC image.
Average pixel intensity is used to normalize among cells.
[0074] iv. Single Cell Fluorescence Microscopy
[0075] In some embodiments, the invention provides measurement of
transporter characteristics at the single-cell level. Single-cell
fluorescence microscopy provides a powerful assay to study rapid
neurotransmitter uptake kinetics from single cells.
[0076] V. Automation
[0077] The inventors further contemplate that all methods disclosed
herein are adaptable to high-throughput formats using robotic fluid
dispensers, multi-well formats and fluorescent plate readers for
the identification of neurotransmitter transport modulators.
Examples are provided in Example 3 and FIGS. 9-11.
[0078] vi. Other Methods
[0079] In addition, uptake and accumulation of the neurotransmitter
may be also characterized by other methods known in the art such as
(a) in vivo inhibition by known agonists and antagonists of the
neurotransmitter transporter; (b) knockout models, where a
particular gene that modulates in or otherwise suspected to be
involved in transport is omitted (for example, DAT knockouts as
described in Giros et al., 1996) (c) slice electrophysiology, in
which particular neurons are identified and subjected to analysis
in situ.
D. Screening For Neurotransmitter Modulators
[0080] Defects in neurotransmitter transporters are associated with
various nervous system disorders including depression, stress
disorders, attention deficit disorder, Parkinson's disease,
anxiety, obesity, several sleep related disorders and certain
neurodegenerative diseases (Edwards, 1993). For example, biogenic
amine transporters which are responsible for inactivation of
dopamine, norepinephrine, serotonin and epinephrine are major
targets for multiple psychoactive substances including cocaine,
amphetamines, methylphenidate (Ritalin.TM.), tricyclic
antidepressants and the SSRIs such as fluoxetine (Prozac.TM.).
However, there is still a need in the art to identify other
modulators of neurotransmitter transporters given the large number
of neurological and psychiatric diseases that are associated with
transporter defects.
[0081] The present invention provides methods for identifying
modulators of the function of neurotransmitter transporters. These
methods may comprise random screening of large libraries of
candidate substances. Alternatively, the methods may be used to
focus on particular classes of compounds selected with an eye
towards structural attributes that are believed to make them more
likely to modulate the function of a particular neurotransmitter
transporter.
[0082] By function, it is meant that one may assay for uptake,
accumulation, or clearance of the neurotransmitter, its analog or
derivative or for some biological aspect of neurotransmitter
release, uptake or clearance. Micro-dialysis and amperometry may be
used to assay transporter finction in vivo (Giros et al., 1996;
Galli et al. 1998).
[0083] To identify a neurotransmitter transporter modulator, one
generally will determine the finction of the neurotransmitter
transporter in the presence and absence of the candidate agent, a
modulator defined as any agent that alters function. For example, a
method generally comprises:
[0084] a) providing a candidate modulator;
[0085] b) contacting the candidate modulator with a cell expressing
a neurotransmitter transporter, or a cell extract or cell membrane
preparation that comprises the neurotransmitter transporter, or a
suitable experimental animal;
[0086] c) measuring one or more characteristics of the transporter,
cell, cell extract or cell membrane preparation, or animal, that
reflects the function or activity of the transporter; and
[0087] d) comparing the characteristic measured in step (c) with
the characteristic of the transporter, cell, cell extract or cell
membrane preparation, or animal in the absence of the candidate
modulator,
[0088] wherein a difference between the measured characteristics
indicates that the candidate modulator is, indeed, a modulator of
the neurotransmitter transporter.
[0089] Comparing the characteristic measured as described in the
steps above includes measurement of uptake, accumulation, binding,
ion dependence, antagonist block, dependence on expression level,
voltage- and Ca.sup.2+-dependence, or clearance of ASP.sup.+ or
other fluorescent substrate that is specifically taken up by the
neurotransmitter transporter.
[0090] Assays may be conducted in cell free systems such as
cellular extracts, cell membrane preparations which may be prepared
by lysing cells, in isolated cells, in cells that express
endogenous a neurotransmitter transporter, in cells that are
genetically engineered to express a neurotransmitter transporter,
in cells that exogenously or endogenously express mutant or
functionally deficient transporters, or in organisms including
transgenic animals or animal models of diseases wherein the disease
is associated with neurotransmitter transporters. Thus, knockouts
for neurotransmitter transporters may be used (Giros et al., 1996;
Sora et al., 2001). It will, of course, be understood that all the
screening methods of the present invention are useful in themselves
notwithstanding the fact that effective candidates may not be
found. The invention provides methods for screening for such
candidates, not solely methods of finding them.
[0091] i. Modulators
[0092] As used herein the term "candidate substance" or "candidate
agent" refers to any molecule that may potentially inhibit or
enhance the activity of a neurotransmitter transporter. The
candidate substance may be a protein or fragment thereof, a small
molecule, or even a nucleic acid molecule. It may prove to be the
case that the most useful pharmacological compounds will be
compounds that are structurally related to the known
neurotransmitter transporter modulators, agonists and antagonists
such as cocaine, amphetamines, monoamine oxidase inhibitors,
imipramine and the like. Using lead compounds to help develop
improved compounds is know as "rational drug design" and includes
not only comparisons with know inhibitors and activators, but
predictions relating to the structure of target molecules.
[0093] The goal of rational drug design is to produce structural
analogs of biologically active polypeptides or target compounds. By
creating such analogs, it is possible to fashion drugs, which are
more active or stable than the natural molecules, which have
different susceptibility to alteration or which may affect the
function of various other molecules. In one approach, one would
generate a three-dimensional structure for a target molecule, or a
fragment thereof. This could be accomplished by x-ray
crystallography, computer modeling or by a combination of both
approaches.
[0094] It also is possible to use antibodies to ascertain the
structure of a target compound activator or inhibitor. In
principle, this approach yields a pharmacore upon which subsequent
drug design can be based. It is possible to bypass protein
crystallography altogether by generating anti-idiotypic antibodies
to a functional, pharmacologically active antibody. As a mirror
image of a mirror image, the binding site of anti-idiotype would be
expected to be an analog of the original antigen. The anti-idiotype
could then be used to identify and isolate peptides from banks of
chemically- or biologically-produced peptides. Selected peptides
would then serve as the pharmacore.
[0095] On the other hand, one may simply acquire, from various
commercial sources, small molecule libraries that are believed to
meet the basic criteria for useful drugs in an effort to "brute
force" the identification of useful compounds. Screening of such
libraries, including combinatorially generated libraries (e.g.,
peptide libraries), is a rapid and efficient way to screen large
number of related (and unrelated) compounds for activity.
Combinatorial approaches also lend themselves to rapid evolution of
potential drugs by the creation of second, third and fourth
generation compounds modeled of active, but otherwise undesirable
compounds.
[0096] Candidate agents may include fragments or parts of
naturally-occurring compounds, or may be found as active
combinations of known compounds, which are otherwise inactive. It
is proposed that compounds isolated from natural sources, such as
animals, bacteria, fungi, plant sources, including leaves and bark,
and marine samples may be assayed as candidates for the presence of
potentially useful pharmaceutical agents. It will be understood
that the pharmaceutical agents to be screened could also be derived
or synthesized from chemical compositions or man-made compounds.
Thus, it is understood that the candidate substance identified by
the present invention may be peptide, polypeptide, polynucleotide,
small molecule inhibitors or any other compounds that may be
designed through rational drug design starting from known
inhibitors or stimulators.
[0097] In addition to the modulating compounds initially
identified, the inventors also contemplate that other sterically
similar compounds may be formulated to mimic the key portions of
the structure of the modulators. Such compounds, which may include
peptidomimetics of peptide modulators, may be used in the same
manner as the initial modulators.
[0098] An inhibitor according to the present invention may be one
which exerts its inhibitory or activating effect upstream,
downstream or directly on the neurotransmitter transporter.
Regardless of the type of inhibitor or activator identified by the
present screening methods, the effect of the inhibition or
activation by such a compound results in a difference as compared
to that observed in the absence of the added candidate
substance.
[0099] ii. In vitro Assays
[0100] A quick, inexpensive and easy assay to run is an in vitro
assay. Such assays generally use isolated molecules, can be run
quickly and in large numbers, thereby increasing the amount of
information obtainable in a short period of time. A variety of
vessels may be used to run the assays, including test tubes,
plates, dishes and other surfaces such as dipsticks or beads.
[0101] One example of a cell free assay in this invention is the
use of cellular extracts that comprise a neurotransmitter, these
may be cell membrane preparations that comprise a neurotransmitter
transporter.
[0102] Another example is a cell-binding assay. While not directly
addressing finction, the ability of a modulator to bind to a target
molecule (in this case the neurotransmitter transporter) in a
specific fashion is strong evidence of a related biological effect.
For example, binding of a molecule to a neurotransmitter
transporter may, in and of itself, be inhibitory, due to steric,
allosteric or charge-charge interactions. The neurotransmitter
transporter protein may be either free in solution, fixed to a
support, expressed in or on the surface of a cell. Either the
neurotransmitter transporter or the compound may be labeled,
thereby permitting determining of binding. Usually, the target will
be the labeled species, decreasing the chance that the labeling
will interfere with or enhance binding. Competitive binding formats
can be performed in which one of the agents is labeled, and one may
measure the amount of free label versus bound label to determine
the effect on binding.
[0103] A technique for high throughput screening of compounds is
described in WO 84/03564. Large numbers of small peptide test
compounds are synthesized on a solid substrate, such as plastic
pins or some other surface. Bound polypeptide is detected by
various methods.
[0104] iii. In cyto Assays
[0105] The present invention also contemplates the screening of
agents for their ability to modulate neurotransmitter transporter
in cells. Various cells and cell lines can be utilized for such
screening assays as long as the cell expresses a neurotransmitter
transporter. This includes cells specifically engineered to
expresses a neurotransmitter transporter. Such cells and nucleic
acid vectors are described in several sections infra as well as
U.S. Pat. Nos. 5,312,734, 5,418,162, and 5,424,185, the contents of
which are all incorporated herein by reference.
[0106] Depending on the assay, culture may be required. The cell is
examined using any of a number of different physiologic assays.
Alternatively, molecular analysis may be performed, for example,
looking at protein expression, mRNA expression (including
differential display of whole cell or polyA RNA) and others.
[0107] iv. In vivo Assays
[0108] In vivo assays involve the use of various animal models,
including transgenic animals that have been engineered to have
specific defects, or carry markers that can be used to measure the
ability of a candidate agent to reach and effect expression of
neurotransmitter transporters in different cells within the
organism. Due to their size, ease of handling, and information on
their physiology and genetic make-up, mice and/or C. elegans are a
preferred embodiment, especially for transgenics. However, other
animals are suitable as well, including rats, rabbits, hamsters,
guinea pigs, gerbils, woodchucks, cats, dogs, sheep, goats, pigs,
cows, horses and monkeys (including chimps, gibbons and baboons).
Assays for modulators may be conducted using an animal model
derived from any of these species.
[0109] In such assays, one or more candidate agents are
administered to an animal, and the ability of the candidate
agent(s) to alter one or more characteristics that are a result of
neurotransmitter finction or activity, as compared to a similar
animal not treated with the candidate agent(s), identifies a
modulator. The characteristics may be any of those discussed above
with regard to the finction of a particular neurotransmitter such
as change in neurotransmission, change in the activity of some
other downstream protein due to a change in neurotransmission, or
instead a broader indication such as behavior of an animal etc.
[0110] The present invention provides methods of screening for
candidate agents that modulate neurotransmitter transporter
finction or activity. In these embodiments, the present invention
is directed to a method for determining the ability of a candidate
agent to modulate neurotransmitter transporter finction, generally
including the steps of: administering a candidate substance to the
animal; and determining the ability of the candidate substance to
change one or more characteristics of the neurotransmitter
transporter.
[0111] Methods for in vivo imaging using ASP.sup.+ are described in
Herrera and Banner (1990), and in Herrera et al., (1990), (both
incorporated herein by reference). In situ methods for analysis of
ASP.sup.+ are described in Pietruck & Ullrich, (1995) and
Rohlicek & Ullrich, (1994), (also incorporated herein by
reference). These methods may be suitably modified with the other
teachings of this specification to perform the in vivo assays.
[0112] Treatment of these animals with test agents will involve the
administration of the agent, in an appropriate form, to the animal.
Administration will be by any route that could be utilized for
clinical or non-clinical purposes, including but not limited to
oral, nasal, buccal, or even topical. Alternatively, administration
may be by parenteral methods such as intratracheal instillation,
bronchial instillation, intradermal, subcutaneous, intramuscular,
intraperitoneal or intravenous injection. Specifically contemplated
routes are systemic intravenous injection, regional administration
via blood or lymph supply, or directly to an affected site.
[0113] Determining the effectiveness of a compound in vivo may
involve a variety of different criteria. Also, measuring toxicity
and dose response can be performed in animals in a more meaningful
fashion than in in vitro or in cyto assays.
F. Vectors for Delivery and Expression of Neurotransmitter
Transporters
[0114] Within certain embodiments, expression vectors are employed
to express a neurotransmitter transporter in a cell, for example,
an DAT, NET, ET, or SERT. The specification provides a description
of transformation of HEK cells to express exogenous NET as one
example infra. Furthermore, U.S. Pat. Nos. 5,312,734, 5,418,162,
and 5,424,185, all incorporated herein by reference, describe
nucleic acids, vectors, and host cells used to express various
neurotransmitter transporters in cells. As will be understood by
one of skill in the art, the invention is not limited to any
particular type of neurotransmitter transporter or cell type and
expression vectors encoding any neurotransmitter transporter can be
used in any cell type. Additionally, as set forth above one may
also use mutant versions, isoforms, and other variants of any
neurotransmitter transporter in the methods of the invention. The
foregoing section provides a general description of how exogenous
expression may be achieved.
[0115] Expression requires that appropriate signals be provided in
the vectors, and which include various regulatory elements, such as
enhancers/promoters from both viral and mammalian sources that
drive expression of the genes of interest in host cells. Elements
designed to optimize messenger RNA stability and translatability in
host cells also are defined. The conditions for the use of a number
of dominant drug selection markers for establishing permanent,
stable cell clones expressing the products are also provided, as is
an element that links expression of the drug selection markers to
expression of the polypeptide.
[0116] i. Regulatory Elements
[0117] Throughout this application, the term "expression construct"
is meant to include any type of genetic construct containing a
nucleic acid coding for a gene product in which part or all of the
nucleic acid encoding sequence is capable of being transcribed and
translated into a polypeptide product. An "expression cassette" is
defined as a nucleic acid encoding a gene product under
transcriptional control of a promoter. A "promoter" refers to a DNA
sequence recognized by the synthetic machinery of the cell, or
introduced synthetic machinery, required to initiate the specific
transcription of a gene. The phrase "under transcriptional control"
means that the promoter is in the correct location and orientation
in relation to the nucleic acid to control RNA polymerase
initiation and expression of the gene.
[0118] The term promoter will be used here to refer to a group of
transcriptional control modules that are clustered around the
initiation site for RNA polymerase II. Much of the thinking about
how promoters are organized derives from analyses of several viral
promoters, including those for the HSV thymidine kinase (tk) and
SV40 early transcription units. These studies, augmented by more
recent work, have shown that promoters are composed of discrete
functional modules, each consisting of approximately 7-20 bp of
DNA, and containing one or more recognition sites for
transcriptional activator or repressor proteins.
[0119] At least one module in each promoter functions to position
the start site for RNA synthesis. The best known example of this is
the TATA box, but in some promoters lacking a TATA box, such as the
promoter for the mammalian terminal deoxynucleotidyl transferase
gene and the promoter for the SV40 late genes, a discrete element
overlying the start site itself helps to fix the place of
initiation.
[0120] Additional promoter elements regulate the frequency of
transcriptional initiation. Typically, these are located in the
region 30-110 bp upstream of the start site, although a number of
promoters have recently been shown to contain finctional elements
downstream of the start site as well. The spacing between promoter
elements frequently is flexible, so that promoter function is
preserved when elements are inverted or moved relative to one
another. In the tk promoter, the spacing between promoter elements
can be increased to 50 bp apart before activity begins to decline.
Depending on the promoter, it appears that individual elements can
function either co-operatively or independently to activate
transcription.
[0121] In certain embodiments, the human cytomegalovirus (CMV)
immediate early gene promoter, the SV40 early promoter, the Rous
sarcoma virus long terminal repeat, rat insulin promoter and
glyceraldehyde-3-phosphate dehydrogenase can be used to obtain
high-level expression of the coding sequence of interest. The use
of other viral or mammalian cellular or bacterial phage promoters
which are well-known in the art to achieve expression of a coding
sequence of interest is contemplated as well, provided that the
levels of expression are sufficient for a given purpose.
[0122] By employing a promoter with well-known properties, the
level and pattern of expression of the protein of interest
following transfection or transformation can be optimized. Further,
selection of a promoter that is regulated in response to specific
physiologic signals can permit inducible expression of the gene
product. Tables 1 and 2 list several regulatory elements that may
be employed, in the context of the present invention, to regulate
the expression of the gene of interest. This list is not intended
to be exhaustive of all the possible elements involved in the
promotion of gene expression but, merely, to be exemplary
thereof.
[0123] Enhancers are genetic elements that increase transcription
from a promoter located at a distant position on the same molecule
of DNA. Enhancers are organized much like promoters. That is, they
are composed of many individual elements, each of which binds to
one or more transcriptional proteins.
[0124] The basic distinction between enhancers and promoters is
operational. An enhancer region as a whole must be able to
stimulate transcription at a distance; this need not be true of a
promoter region or its component elements. On the other hand, a
promoter must have one or more elements that direct initiation of
RNA synthesis at a particular site and in a particular orientation,
whereas enhancers lack these specificities. Promoters and enhancers
are often overlapping and contiguous, often seeming to have a very
similar modular organization.
[0125] Below is a list of viral promoters, cellular
promoters/enhancers and inducible promoters/enhancers that could be
used in combination with the nucleic acid encoding a gene of
interest in an expression construct (Table 1 and Table 2).
Additionally, any promoter/enhancer combination (as per the
Eukaryotic Promoter Data Base EPDB) could also be used to drive
expression of the gene. Eukaryotic cells can support cytoplasmic
transcription from certain bacterial promoters if the appropriate
bacterial polymerase is provided, either as part of the delivery
complex or as an additional genetic expression construct.
1TABLE 1 Promoter and/or Enhancer Promoter/Enhancer References
Immunoglobulin Heavy Chain Banerji et al., 1983; Gilles et al.,
1983; Grosschedl et al., 1985; Atchinson et al., 1986, 1987; Imler
et al., 1987; Weinberger et al., 1984; Kiledjian et al., 1988;
Porton et al.; 1990 Immunoglobulin Light Chain Queen et al., 1983;
Picard et al., 1984 T-Cell Receptor Luria et al., 1987; Winoto et
al., 1989; Redondo et al.; 1990 HLA DQ a and/or DQ .beta. Sullivan
et al., 1987 .beta.-Interferon Goodbourn et al., 1986; Fujita et
al., 1987; Goodbourn et al., 1988 Interleukin-2 Greene et al., 1989
lnterleukin-2 Receptor Greene et al., 1989; Lin et al., 1990 MHC
Class II 5 Koch et al., 1989 MHC Class II HLA-Dra Sherman et al.,
1989 .beta.-Actin Kawamoto et al., 1988; Ng et al.; 1989 Muscle
Creatine Kinase (MCK) Jaynes et al., 1988; Horlick et al., 1989;
Johnson et al., 1989 Prealbumin (Transthyretin) Costa et al., 1988
Elastase I Ornitz et al., 1987 Metallothionein (MTII) Karin et al.,
1987; Culotta et al., 1989 Collagenase Pinkert et al., 1987; Angel
et al., 1987 Albumin Pinkert et al., 1987; Tronche et al., 1989,
1990 .alpha.-Fetoprotein Godbout et al., 1988; Campere et al., 1989
.gamma.-Globin Bodine et al., 1987; Perez-Stable et al., 1990
.beta.-Globin Trudel et al., 1987 c-fos Cohen et al., 1987 c-HA-ras
Triesman, 1986; Deschamps et al., 1985 Insulin Edlund et al., 1985
Neural Cell Adhesion Molecule Hirsch et al., 1990 (NCAM)
.alpha..sub.1-Antitrypsin Latimer et al., 1990 H2B (TH2B) Histone
Hwang et al., 1990 Mouse and/or Type I Collagen Ripe et al., 1989
Glucose-Regulated Proteins Chang et al., 1989 (GRP94 and GRP78) Rat
Growth Hormone Larsen et al., 1986 Human Serum Amyloid A (SAA)
Edbrooke et al., 1989 Troponin I (TN I) Yutzey et al., 1989
Platelet-Derived Growth Factor Pech et al., 1989 (PDGF) Duchenne
Muscular Dystrophy Klamut et al., 1990 SV40 Banerji et al., 1981;
Moreau et al., 1981; Sleigh et al., 1985; Firak et al., 1986; Herr
et al., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wang et
al., 1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al.,
1988 Polyoma Swartzendruber et al., 1975; Vasseur et al., 1980;
Katinka et al., 1980, 1981; Tyndell et al., 1981; Dandolo et al.,
1983; de Villiers et al., 1984; Hen et al., 1986; Satake et al.,
1988; Campbell and/or Villarreal, 1988 Retroviruses Kriegler et
al., 1982, 1983; Levinson et al., 1982; Kriegler et al., 1983,
1984a, b, 1988; Bosze et al., 1986; Miksicek et al., 1986; Celander
et al., 1987; Thiesen et al., 1988; Celander et al., 1988; Choi et
al., 1988; Reisman et al., 1989 Papilloma Virus Campo et al., 1983;
Lusky et al., 1983; Spandidos and/or Wilkie, 1983; Spalholz et al.,
1985; Lusky et al., 1986; Cripe et al., 1987; Gloss et al., 1987;
Hirochika et al., 1987; Stephens et al., 1987 Hepatitis B Virus
Bulla et al., 1986; Jameel et al., 1986; Shaul et al., 1987;
Spandau et al., 1988; Vannice et al., 1988 Human Immunodeficiency
Virus Muesing et al., 1987; Hauber et al., 1988; Jakobovits et al.,
1988; Feng et al., 1988; Takebe et al., 1988; Rosen et al., 1988;
Berkhout et al., 1989; Laspia et al., 1989; Sharp et al., 1989;
Braddock et al., 1989 Cytomegalovirus (CMV) Weber et al., 1984;
Boshart et al., 1985; Foecking et al., 1986 Gibbon Ape Leukemia
Virus Holbrook et al., 1987; Quinn et al., 1989
[0126]
2TABLE 2 Inducible Elements Element Inducer References MT II
Phorbol Ester (TFA) Palmiter et al., 1982; Heavy metals Haslinger
et al., 1985; Searle et al., 1985; Stuart et al., 1985; Imagawa et
al., 1987, Karin et al., 1987; Angel et al., 1987b; McNeall et al.,
1989 MMTV (mouse Glucocorticoids Huang et al., 1981; Lee mammary et
al., 1981; Majors et al., tumor virus) 1983; Chandler et al., 1983;
Lee et al., 1984; Ponta et al., 1985; Sakai et al., 1988
.beta.-Interferon Poly(rI)x Tavernier et al., 1983 Poly(rc)
Adenovirus 5 E2 E1A Imperiale et al., 1984 Collagenase Phorbol
Ester (TPA) Angel et al., 1987a Stromelysin Phorbol Ester (TPA)
Angel et al., 1987b SV40 Phorbol Ester (TPA) Angel et al., 1987b
Murine MX Gene Interferon, Newcastle Hug et al., 1988 Disease Virus
GRP78 Gene A23187 Resendez et al., 1988 .alpha.-2-Macroglobulin
IL-6 Kunz et al., 1989 Vimentin Serum Rittling et al., 1989 MHC
Class I Gene Interferon Blanar et al., 1989 H-2.kappa.b E1A, SV40
Large T Taylor et al., 1989, 1990a, HSP70 Antigen 1990b Proliferin
Phorbol Ester-TPA Mordacq et al., 1989 Tumor Necrosis PMA Hensel et
al., 1989 Factor .alpha. Thyroid Stimulating Thyroid Hormone
Chatterjee et al., 1989 Hormone .alpha. Gene
[0127] Where a cDNA insert is employed, one will typically desire
to include a polyadenylation signal to effect proper
polyadenylation of the gene transcript. The nature of the
polyadenylation signal is not believed to be crucial to the
successful practice of the invention, and any such sequence may be
employed such as human growth hormone and SV40 polyadenylation
signals. Also contemplated as an element of the expression cassette
is a terminator. These elements can serve to enhance message levels
and to minimize read through from the cassette into other
sequences.
[0128] ii. Selectable Markers
[0129] In certain embodiments of the invention, the cells contain
nucleic acid constructs encoding a neurotransmitter transporter may
be identified in vitro or in vivo by including a marker in the
expression construct. Such markers would confer an identifiable
change to the cell permitting easy identification of cells
containing the expression construct. Usually the inclusion of a
drug selection marker aids in cloning and in the selection of
transformants, for example, genes that confer resistance to
neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol
are useful selectable markers. Alternatively, enzymes such as
herpes simplex virus thymidine kinase (tk) or chloramphenicol
acetyltransferase (CAT) may be employed. Immunologic markers also
can be employed. The selectable marker employed is not believed to
be important, so long as it is capable of being expressed
simultaneously with the nucleic acid encoding a gene product.
Further examples of selectable markers are well known to one of
skill in the art.
[0130] iii. Polyadenylation Signals
[0131] In expression, one will typically include a polyadenylation
signal to effect proper polyadenylation of the transcript. The
nature of the polyadenylation signal is not believed to be crucial
to the successful practice of the invention, and/or any such
sequence may be employed. Preferred embodiments include the SV40
polyadenylation signal and/or the bovine growth hormone
polyadenylation signal, convenient and/or known to function well in
various target cells. Also contemplated as an element of the
expression cassette is a transcriptional termination site. These
elements can serve to enhance message levels and/or to minimize
read through from the cassette into other sequences.
[0132] iv. Vectors
[0133] The term "vector" is used to refer to a carrier nucleic acid
molecule into which a nucleic acid sequence can be inserted for
introduction into a cell where it can be replicated. A nucleic acid
sequence can be "exogenous," which means that it is foreign to the
cell into which the vector is being introduced or that the sequence
is homologous to a sequence in the cell but in a position within
the host cell nucleic acid in which the sequence is ordinarily not
found. Vectors include plasmids, cosmids, viruses (bacteriophage,
animal viruses, and plant viruses), and artificial chromosomes
(e.g., YACs). One of skill in the art would be well equipped to
construct a vector through standard recombinant techniques, which
are described in Sambrook et al. (1989) and Ausubel et al. (1994),
both incorporated herein by reference.
[0134] The term "expression vector" refers to a vector containing a
nucleic acid sequence coding for at least part of a gene product
capable of being transcribed. In some cases, RNA molecules are then
translated into a protein, polypeptide, or peptide. In other cases,
these sequences are not translated, for example, in the production
of antisense molecules or ribozymes. Expression vectors can contain
a variety of "control sequences," which refer to nucleic acid
sequences necessary for the transcription and possibly translation
of an operably linked coding sequence in a particular host
organism. In addition to control sequences that govern
transcription and translation, vectors and expression vectors may
contain nucleic acid sequences that serve other functions as well
and are described infra.
[0135] V. Delivery of Expression Vectors
[0136] There are a number of ways in which expression vectors may
be introduced into cells. In certain embodiments of the invention,
the expression construct comprises a virus or engineered construct
derived from a viral genome. The ability of certain viruses to
enter cells via receptor-mediated endocytosis, to integrate into
host cell genome and express viral genes stably and efficiently
have made them attractive candidates for the transfer of foreign
genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein,
1988; Baichwal and Sugden, 1986; Temin, 1986). The first viruses
used as gene vectors were DNA viruses including the papovaviruses
(simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway,
1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988;
Baichwal and Sugden, 1986). These have a relatively low capacity
for foreign DNA sequences and have a restricted host spectrum.
Furthermore, their oncogenic potential and cytopathic effects in
permissive cells raise safety concerns. They can accommodate only
up to 8 kB of foreign genetic material but can be readily
introduced in a variety of cell lines and laboratory animals
(Nicolas and Rubenstein, 1988; Temin, 1986).
[0137] a. Adenovirus
[0138] One of the methods for in vivo delivery involves the use of
an adenovirus expression vector. "Adenovirus expression vector" is
meant to include those constructs containing adenovirus sequences
sufficient to (a) support packaging of the construct and (b) to
express an antisense polynucleotide that has been cloned therein.
In this context, expression does not require that the gene product
be synthesized.
[0139] The expression vector comprises a genetically engineered
form of adenovirus. Knowledge of the genetic organization of
adenovirus, a 36 kB, linear, double-stranded DNA virus, allows
substitution of large pieces of adenoviral DNA with foreign
sequences up to 7 kB (Grunhaus and Horwitz, 1992). In contrast to
retrovirus, the adenoviral infection of host cells does not result
in chromosomal integration because adenoviral DNA can replicate in
an episomal manner without potential genotoxicity. Also,
adenoviruses are structurally stable, and no genome rearrangement
has been detected after extensive amplification. Adenovirus can
infect virtually all epithelial cells regardless of their cell
cycle stage. So far, adenoviral infection appears to be linked only
to mild disease such as acute respiratory disease in humans.
[0140] Adenovirus is particularly suitable for use as a gene
transfer vector because of its mid-sized genome, ease of
manipulation, high titer, wide target cell range and high
infectivity. Both ends of the viral genome contain 100-200 base
pair inverted repeats (ITRs), which are cis elements necessary for
viral DNA replication and packaging. The early (E) and late (L)
regions of the genome contain different transcription units that
are divided by the onset of viral DNA replication. The El region
(ElA and E1B) encodes proteins responsible for the regulation of
transcription of the viral genome and a few cellular genes. The
expression of the E2 region (E2A and E2B) results in the synthesis
of the proteins for viral DNA replication. These proteins are
involved in DNA replication, late gene expression and host cell
shut-off(Renan, 1990). The products of the late genes, including
the majority of the viral capsid proteins, are expressed only after
significant processing of a single primary transcript issued by the
major late promoter (MLP). The MLP (located at 16.8 m.u.) is
particularly efficient during the late phase of infection, and all
the mRNA's issued from this promoter possess a 5'-tripartite leader
(TPL) sequence which makes them preferred mRNA's for
translation.
[0141] In a current system, recombinant adenovirus is generated
from homologous recombination between shuttle vector and provirus
vector. Due to the possible recombination between two proviral
vectors, wild-type adenovirus may be generated from this process.
Therefore, it is critical to isolate a single clone of virus from
an individual plaque and examine its genomic structure.
[0142] Generation and propagation of the current adenovirus
vectors, which are replication deficient, depend on a unique helper
cell line, designated 293, which was transformed from human
embryonic kidney cells by AdS DNA fragments and constitutively
expresses El proteins (Graham et al., 1977). Since the E3 region is
dispensable from the adenovirus genome (Jones and Shenk, 1978), the
current adenovirus vectors, with the help of 293 cells, carry
foreign DNA in either the El, the D3 or both regions (Graham and
Prevec, 1991). In nature, adenovirus can package approximately 105%
of the wild-type genome (Ghosh-Choudhury et al., 1987), providing
capacity for about 2 extra kb of DNA. Combined with the
approximately 5.5 kb of DNA that is replaceable in the E1 and E3
regions, the maximum capacity of the current adenovirus vector is
under 7.5 kb, or about 15% of the total length of the vector. More
than 80% of the adenovirus viral genome remains in the vector
backbone and is the source of vector-borne cytotoxicity. Also, the
replication deficiency of the E1-deleted virus is incomplete. For
example, leakage of viral gene expression has been observed with
the currently available vectors at high multiplicities of infection
(MO1) (Mulligan, 1993).
[0143] Helper cell lines may be derived from human cells such as
human embryonic kidney cells, muscle cells, hematopoietic cells or
other human embryonic mesenchymal or epithelial cells.
Alternatively, the helper cells may be derived from the cells of
other mammalian species that are permissive for human adenovirus.
Such cells include, e.g., Vero cells or other monkey embryonic
mesenchymal or epithelial cells. As stated above, the preferred
helper cell line is 293.
[0144] Racher et al. (1995) disclosed improved methods for
culturing 293 cells and propagating adenovirus. In one format,
natural cell aggregates are grown by inoculating individual cells
into 1 liter siliconized spinner flasks (Techne, Cambridge, UK)
containing 100-200 ml of medium. Following stirring at 40 rpm, the
cell viability is estimated with trypan blue. In another format,
Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/l) is
employed as follows. A cell innoculum, resuspended in 5 ml of
medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer
flask and left stationary, with occasional agitation, for 1 to 4 h.
The medium is then replaced with 50 ml of fresh medium and shaking
initiated. For virus production, cells are allowed to grow to about
80% confluence, after which time the medium is replaced (to 25% of
the final volume) and adenovirus added at an MOI of 0.05. Cultures
are left stationary overnight, following which the volume is
increased to 100% and shaking commenced for another 72 h.
[0145] Other than the requirement that the adenovirus vector be
replication defective, or at least conditionally defective, the
nature of the adenovirus vector is not believed to be crucial to
the successful practice of the invention. The adenovirus may be of
any of the 42 different known serotypes or subgroups A-F.
Adenovirus type 5 of subgroup C is the preferred starting material
in order to obtain the conditional replication-defective adenovirus
vector for use in the present invention. This is because Adenovirus
type 5 is a human adenovirus about which a great deal of
biochemical and genetic information is known, and it has
historically been used for most constructions employing adenovirus
as a vector.
[0146] As stated above, the typical vector according to the present
invention is replication defective and will not have an adenovirus
E1 region. Thus, it will be most convenient to introduce the
polynucleotide encoding the gene of interest at the position from
which the E1-coding sequences have been removed. However, the
position of insertion of the construct within the adenovirus
sequences is not critical to the invention. The polynucleotide
encoding the gene of interest may also be inserted in lieu of the
deleted E3 region in E3 replacement vectors, as described by
Karlsson et al. (1986), or in the E4 region where a helper cell
line or helper virus complements the E4 defect.
[0147] Adenovirus is easy to grow and manipulate and exhibits broad
host range in vitro and in vivo. This group of viruses can be
obtained in high titers, e.g., 10.sup.9-10.sup.12 plaque-forming
units per ml, and they are highly infective. The life cycle of
adenovirus does not require integration into the host cell genome.
The foreign genes delivered by adenovirus vectors are episomal and,
therefore, have low genotoxicity to host cells. No side effects
have been reported in studies of vaccination with wild-type
adenovirus (Couch et al., 1963; Top et al., 1971), demonstrating
their safety and therapeutic potential as in vivo gene transfer
vectors.
[0148] Adenovirus vectors have been used in eukaryotic gene
expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and
vaccine development (Grunhaus & Horwitz, 1992; Graham and
Prevec, 1991). Recently, animal studies suggested that recombinant
adenovirus could be used for gene therapy (Stratford-Perricaudet
& Perricaudet, 1991; Stratford-Perricaudet et al., 1990; Rich
et al., 1993). Studies in administering recombinant adenovirus to
different tissues include trachea instillation (Rosenfeld et al,
1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,
1993), peripheral intravenous injections (Herz and Gerard, 1993)
and stereotactic inoculation into the brain (Le Gal La Salle et
al., 1993).
[0149] b. Retrovirus
[0150] The retroviruses are a group of single-stranded RNA viruses
characterized by an ability to convert their RNA to double-stranded
DNA in infected cells by a process of reverse-transcription
(Coffin, 1990). The resulting DNA then stably integrates into
cellular chromosomes as a provirus and directs synthesis of viral
proteins. The integration results in the retention of the viral
gene sequences in the recipient cell and its descendants. The
retroviral genome contains three genes, gag, pol, and env that code
for capsid proteins, polymerase enzyme, and envelope components,
respectively. A sequence found upstream from the gag gene contains
a signal for packaging of the genome into virions. Two long
terminal repeat (LTR) sequences are present at the 5' and 3' ends
of the viral genome. These contain strong promoter and enhancer
sequences and are also required for integration in the host cell
genome (Coffin, 1990).
[0151] In order to construct a retroviral vector, a nucleic acid
encoding a gene of interest is inserted into the viral genome in
the place of certain viral sequences to produce a virus that is
replication-defective. In order to produce virions, a packaging
cell line containing the gag, pol, and env genes but without the
LTR and packaging components is constructed (Mann et al., 1983).
When a recombinant plasmid containing a cDNA, together with the
retroviral LTR and packaging sequences is introduced into this cell
line (by calcium phosphate precipitation for example), the
packaging sequence allows the RNA transcript of the recombinant
plasmid to be packaged into viral particles, which are then
secreted into the culture media (Nicolas and Rubenstein, 1988;
Temin, 1986; Mann et al., 1983). The media containing the
recombinant retroviruses is then collected, optionally
concentrated, and used for gene transfer. Retroviral vectors are
able to infect a broad variety of cell types. However, integration
and stable expression require the division of host cells (Paskind
et al., 1975).
[0152] A novel approach designed to allow specific targeting of
retrovirus vectors was recently developed based on the chemical
modification of a retrovirus by the chemical addition of lactose
residues to the viral envelope. This modification could permit the
specific infection of hepatocytes via sialoglycoprotein
receptors.
[0153] A different approach to targeting of recombinant
retroviruses was designed in which biotinylated antibodies against
a retroviral envelope protein and against a specific cell receptor
were used. The antibodies were coupled via the biotin components by
using streptavidin (Roux et al., 1989). Using antibodies against
major histocompatibility complex class I and class II antigens,
they demonstrated the infection of a variety of human cells that
bore those surface antigens with an ecotropic virus in vitro (Roux
et al., 1989).
[0154] There are certain limitations to the use of retrovirus
vectors in all aspects of the present invention. For example,
retrovirus vectors usually integrate into random sites in the cell
genome. This can lead to insertional mutagenesis through the
interruption of host genes or through the insertion of viral
regulatory sequences that can interfere with the function of
flanking genes (Varmus et al., 1981). Another concern with the use
of defective retrovirus vectors is the potential appearance of
wild-type replication-competent virus in the packaging cells. This
can result from recombination events in which the intact- sequence
from the recombinant virus inserts upstream from the gag, pol, env
sequence integrated in the host cell genome. However, new packaging
cell lines are now available that should greatly decrease the
likelihood of recombination (Markowitz et al., 1988; Hersdorffer et
al., 1990).
[0155] c. Adeno-Associated Viruses
[0156] Adeno-associated virus (AAV) is an attractive virus for
delivering foreign genes to mammalian cells or subjects (Ridgeway,
1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984). AAV
utilizes a linear, single-stranded DNA of about 4700 base pairs.
Inverted terminal repeats flank the genome. Two genes are present
within the genome, giving rise to a number of distinct gene
products. The first, the cap gene, produces three different virion
proteins (VP), designated VP-1, VP-2 and VP-3. The second, the rep
gene, encodes four non-structural proteins (NS). One or more of
these rep gene products is responsible for transactivating AAV
transcription. The sequence of AAV is provided by U.S. Pat. No.
5,252,479 (entire text of which is specifically incorporated herein
by reference).
[0157] The three promoters in AAV are designated by their location,
in map units, in the genome. These are, from left to right, p5, pl9
and p40. Transcription gives rise to six transcripts, two initiated
at each of three promoters, with one of each pair being spliced.
The splice site, derived from map units 42-46, is the same for each
transcript. The four non-structural proteins apparently are derived
from the longer of the transcripts, and three virion proteins all
arise from the smallest transcript.
[0158] AAV is not associated with any pathologic state in humans.
Interestingly, for efficient replication, AAV requires "helping"
functions from viruses such as herpes simplex virus I and II,
cytomegalovirus, pseudorabies virus and, of course, adenovirus. The
best characterized of the helpers is adenovirus, and many "early"
functions for this virus have been shown to assist with AAV
replication. Low level expression of AAV rep proteins is believed
to hold AAV structural expression in check, and helper virus
infection is thought to remove this block.
[0159] The terminal repeats of the AAV vector of the present
invention can be obtained by restriction endonuclease digestion of
AAV or a plasmid such as p201, which contains a modified AAV genome
(Samulski et al., 1987). Alternatively, the terminal repeats may be
obtained by other methods known to the skilled artisan, including
but not limited to chemical or enzymatic synthesis of the terminal
repeats based upon the published sequence of AAV. The ordinarily
skilled artisan can determine, by well-known methods such as
deletion analysis, the minimum sequence or part of the AAV ITRs
which is required to allow function, i.e., stable and site-specific
integration. The ordinarily skilled artisan also can determine
which minor modifications of the sequence can be tolerated while
maintaining the ability of the terminal repeats to direct stable,
site-specific integration.
[0160] d. Other Viruses
[0161] Other viral vectors may be employed as expression constructs
in the present invention. Vectors derived from viruses such as
vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar
et a., 1988) and herpesviruses may be employed. They offer several
attractive features for various mammalian cells (Friedmann, 1989;
Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988;
Horwich et al., 1990).
[0162] With the recent recognition of defective hepatitis B
viruses, new insight was gained into the structure-function
relationship of different viral sequences. In vitro studies showed
that the virus could retain the ability for helper-dependent
packaging and reverse transcription despite the deletion of up to
80% of its genome (Horwich et al., 1990). This suggested that large
portions of the genome could be replaced with foreign genetic
material. The hepatotropism and persistence (integration) were
particularly attractive properties for liver-directed gene
transfer. Chang et al. (1989, 1991) recently introduced the
chloramphenicol acetyltransferase (CAT) gene into duck hepatitis B
virus genome in the place of the polymerase, surface, and
pre-surface coding sequences. It was co-transfected with wild-type
virus into an avian hepatoma cell line. Culture media containing
high titers of the recombinant virus were used to infect primary
duckling hepatocytes. Stable CAT gene expression was detected for
at least 24 days after transfection (Chang et al., 1991).
[0163] e. Non-Viral Methods
[0164] Several non-viral methods for the transfer of expression
constructs into mammalian cells also are contemplated by the
present invention. These include DEAE-dextran (Gopal, 1985),
electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984),
direct microinjection (Harland and Weintraub, 1985),
lipofectamine-DNA complexes, cell sonication (Fechheimer et al.,
1987), and receptor-mediated transfection (Wu and Wu, 1987; Wu and
Wu, 1988).
[0165] In yet another embodiment of the invention, the expression
construct may simply consist of naked recombinant DNA or plasmids.
Transfer of the construct may be performed by any of the methods
mentioned above which physically or chemically permeabilize the
cell membrane. This is particularly applicable for transfer in
vitro but it may be applied to in vivo use as well. Dubensky et al.
(1984) successfully injected polyomavirus DNA in the form of
calcium phosphate precipitates into liver and spleen of adult and
newborn mice demonstrating active viral replication and acute
infection. Benvenisty and Neshif (1986) also demonstrated that
direct intraperitoneal injection of calcium phosphate-precipitated
plasmids results in expression of the transfected genes. It is
envisioned that DNA encoding a gene of interest may also be
transferred in a similar manner in vivo and express the gene
product.
[0166] f. Liposomes
[0167] In a further embodiment of the invention, the expression
construct may be entrapped in a liposome. Liposomes are vesicular
structures characterized by a phospholipid bilayer membrane and an
inner aqueous medium. Multilamellar liposomes have multiple lipid
layers separated by aqueous medium. They form spontaneously when
phospholipids are suspended in an excess of aqueous solution. The
lipid components undergo self-rearrangement before the formation of
closed structures and entrap water and dissolved solutes between
the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated
are Lipofectamine-DNA complexes.
[0168] Liposome-mediated nucleic acid delivery and expression of
foreign DNA in vitro has been very successful. Wong et al. (1980)
demonstrated the feasibility of liposome-mediated delivery and
expression of foreign DNA in cultured chick embryo, HeLa and
hepatoma cells. Nicolau et al. (1987) accomplished successful
liposome-mediated gene transfer in rats after intravenous
injection.
[0169] In certain embodiments of the invention, the liposome may be
complexed with a hemagglutinating virus (HVJ). This has been shown
to facilitate fusion with the cell membrane and promote cell entry
of liposome-encapsulated DNA (Kaneda et al., 1989). In other
embodiments, the liposome may be complexed or employed in
conjunction with nuclear non-histone chromosomal proteins (HMG-1)
(Kato et al., 1991). In yet further embodiments, the liposome may
be complexed or employed in conjunction with both HVJ and HMG-1. In
that such expression constructs have been successfully employed in
transfer and expression of nucleic acid in vitro and in vivo, then
they are applicable for the present invention. Where a bacterial
promoter is employed in the DNA construct, it also will be
desirable to include within the liposome an appropriate bacterial
polymerase.
[0170] Other expression constructs which can be employed to deliver
a nucleic acid encoding a particular gene into cells are
receptor-mediated delivery vehicles. These take advantage of the
selective uptake of macromolecules by receptor-mediated endocytosis
in almost all eukaryotic cells. Because of the cell type-specific
distribution of various receptors, the delivery can be highly
specific (Wu and Wu, 1993).
[0171] Receptor-mediated gene targeting vehicles generally consist
of two components: a cell receptor-specific ligand and a
DNA-binding agent. Several ligands have been used for
receptor-mediated gene transfer. The most extensively characterized
ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and
transferrin (Wagner et al., 1990). Recently, a synthetic
neoglycoprotein, which recognizes the same receptor as ASOR, has
been used as a gene delivery vehicle (Ferkol et al., 1993; Perales
et al., 1994) and epidermal growth factor (EGF) has also been used
to deliver genes to squamous carcinoma cells (Myers, EPO 0 273
085).
[0172] In other embodiments, the delivery vehicle may comprise a
ligand and a liposome. For example, Nicolau et al. (1987) employed
lactosyl-ceramide, a galactose-terminal asialganglioside,
incorporated into liposomes and observed an increase in the uptake
of the insulin gene by hepatocytes. Thus, it is feasible that a
nucleic acid encoding a particular gene also may be specifically
delivered into a cell type by any number of receptor-ligand systems
with or without liposomes. For example, epidermal growth factor
(EGF) may be used as the receptor for mediated delivery of a
nucleic acid into cells that exhibit upregulation of EGF receptor.
Mannose can be used to target the mannose receptor on liver
cells.
G. Pharmaceutical Formulations
[0173] As the present invention provides clinical methods for the
treatment of neurological diseases using the modulators of
neurotransmitter transporters identified by the screening methods
of the invention, it will be necessary to prepare pharmaceutical
compositions comprising the therapeutic modulatory agent(s) in a
form appropriate for the intended application. Generally, this will
entail preparing compositions that are essentially free of
pyrogens, as well as other impurities that could be harmful to
humans or animals.
[0174] One will generally desire to employ appropriate salts and
buffers. Aqueous compositions of the present invention comprise an
effective amount of the neurotransmitter transporter modulator
dissolved or dispersed in a pharmaceutically acceptable carrier or
aqueous medium. The phrase "pharmaceutically or pharmacologically
acceptable" refer to molecular entities and compositions that do
not produce adverse, allergic, or other untoward reactions when
administered to an animal or a human. As used herein,
"pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents and the like. The
use of such media and agents for pharmaceutically active substances
is well know in the art. Supplementary active ingredients also can
be incorporated into the compositions.
[0175] Administration of these compositions according to the
present invention will be via any common route so long as the
target tissue is available via that route. This includes
administration may be by systemic or parenteral methods including
intravenous injection, intracerebral, intradermal, subcutaneous,
intramuscular, intraperitoneal methods. Direct administration by
local injection into the site of disease is also contemplated.
Depending on the nature of the modulator administration may also be
via oral, nasal, buccal, rectal, vaginal or topical. Such
compositions would normally be administered as pharmaceutically
acceptable compositions, described supra.
[0176] Solutions of the active compounds as free base or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0177] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), suitable mixtures thereof, and vegetable
oils. The proper fluidity can be maintained, for example, by the
use of a coating, such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. The prevention of the action of microorganisms can be
brought about by various antibacterial an antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal,
and the like. In many cases, it will be preferable to include
isotonic agents, for example, sugars or sodium chloride. Prolonged
absorption of the injectable compositions can be brought about by
the use in the compositions of agents delaying absorption, for
example, aluminum monostearate and gelatin.
[0178] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0179] The compositions of the present invention may be formulated
in a neutral or salt form. Pharmaceutically-acceptable salts
include the acid addition salts (formed with the free amino groups
of the protein) and which are formed with inorganic acids such as,
for example, hydrochloric or phosphoric acids, or such organic
acids as acetic, oxalic, tartaric, mandelic, and the like. Salts
formed with the free carboxyl groups can also be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the
like.
[0180] The composition may be formulated as a "unit dose." For
example, one unit dose could be dissolved in 1 ml of isotonic NaCl
solution and either added to 1000 ml of hypodermoclysis fluid or
injected at the proposed site of infusion, (see for example,
"Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038
and 1570-1580). Some variation in dosage will necessarily occur
depending on the condition of the subject being treated. The person
responsible for administration will, in any event, determine the
appropriate dose for the individual subject. Moreover, for human
administration, preparations should meet sterility, pyrogenicity,
general safety and purity standards as required by FDA Office of
Biologics standards.
H. EXAMPLES
[0181] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
[0182] Materials And Methods
[0183] Cell Culture
[0184] HEK-293 cells were maintained in DMEM with 10% FBS (v/v), 2
mM glutamine, 100 I.U./mL penicillin and 100 .mu.g/mL streptomycin
(Gibco). The human norepinephrine transporter (HEK-hNET) and human
serotonin transporter (HEK-hSERT) stable cells line were previously
described (Galli et al., 1995; Ramamoorthy, 1998). The human
dopamine transporter (HEK-hDAT) cells were also used.
[0185] Radiolabled Transport Assay
[0186] All accumulation studies were performed at room temperature
(22.degree. C.) unless otherwise indicated. HEK-hNET cells were
plated on poly-L-lysine coated 24-well tissue culture plates at
10.sup.5 cells per well three days prior to performing transport
assays. The cells were at approximately 90% confluence on the third
day. The medium was removed by aspiration. Cells were then
pre-incubated for 10 min in Krebs-Ringer's-Hepes (KRH: in mM 130
NaCl, 1.3 KCl, 2.2 CaCl.sub.2, 1.2 MgSO.sub.4, 1.2
KH.sub.2PO.sub.4, 10 Hepes, and 1.8 g/L glucose, pH 7.4) medium
with or without 10 .mu.M desipramine. Desipramine, a specific NET
blocker, was used to establish nonspecific activity in hNET cells.
Pargyline (10 .mu.M) and ascorbic acid (10 .mu.M) were added to
prevent metabolism and oxidation of NE, respectively. The assay
mixture was aspirated after 10 minutes and cells were washed three
times with 4.degree. C. KRH buffer. Accumulated [.sup.3H]NE was
determined by liquid scintillation of 1% (w/v) sodium
dodecylsulphate solubilized cells.
[0187] Primary Tissue Culture
[0188] SCG neurons were dissociated by trituration followed by
digestion with 0.25% trypsin and 0.3% collagenase. Non-neuronal
cells were removed by preplating on uncoated, Falcon 60 mm plates.
Neurons were cultured on poly-L-ornithine/laminin/poly D-lysine
coated MatTek Dishes at a density of 3000-4000 cells/well in
F-14.sup.+ media containing 5% fetal calf serum, 2 mM L-glutamine,
60 ng/ml progesterone, 16 mg/ml putrescine, 400 ng/ml L-thyroxine,
38 ng/ml sodium selenite, 340 ng/ml tri-iodothyroxine, 5 mg/ml
insulin, penicillin/streptomycin, 10 .mu.M fluorodeoxyuridine and
20 ng/ml NGF. The neurons were maintained for 3-5 days in the
presence of NGF before use.
[0189] Microscopy
[0190] HEK-hNET cells were plated on 35 mm glass bottom
Petri-dishes (MatTek, Ashland, Mass.) coated with poly-L-lysine
three days prior to experimentation. The culture medium was
aspirated, cells were immediately mounted on a Zeiss 410 confocal
microscope and the microscope was focused on the center of the
monolayer of cells. During the confocal measurment cells remain
without buffer for approximately thirty seconds. Background
autoflourescence was established by collecting images for ten
seconds prior to the addition of KRH (see Radiolabeled
Transport),1.8 mg/L Glucose, 10 .mu.M ascorbic acid, 10 .mu.M
pargalyine, 10 .mu.M tropolone (Sigma, Boulder CO), and ASP.sup.+.
The argon laser was tuned to 488 nm; the emitted light was filtered
with a 580-630 nm band pass filter (.lambda..sub.max=610 nm).
ASP.sup.+ has a large Stoke shift between excitation
(.lambda..sub.max=488 nrn) and emission maxima
(.lambda..sub.max=610 nm). The substantial red shift can be
exploited to reduce background autofluorescence produced in the
absence of substrate. The gain (contrast), offset (brightness) for
the photomultiplier tube (PMT) was set to avoid detector saturation
at the highest ASP.sup.+ concentration used in the transport. The
effects of photobleaching on ASP.sup.+ accumulation were determined
by examining the rate of ASP.sup.+ fluorescence at various
acquisition rates. Acquisition rates greater than 0.3 Hz degraded
ASP.sup.+ , and the inventors set the fastest test acquisition rate
at 12 Hz (80 msec/image).
[0191] Fluorescence Anisotropy Measurements
[0192] To evaluate ASP.sup.+ binding to the surface membranes,
HEK-hNET cells were exposed to 2 .mu.M ASP.sup.+ with horizontal
polarizer (FIG. 5C), with the polarizer rapidly switching to the
vertical position. Cells were imaged with alternating polarizations
for 3 minutes to measure light intensity in the horizontal
(I.sub.h) and vertical (I.sub.v) positions in order to calculate
the anisotropy ratio, r=(I.sub.v-gI.sub.h)/(I.sub.v+2 g I.sub.h).
The factor g was determined by using a half wave plate as described
by Blackman et al. (1996). In this formulation, r=0.4 implies an
immobile source (Smith et al., 1999). Surface anisotropy was
measured from the cell circumference, taken as 1 pixel width.
Cytosolic anisotropy was measured near the center of the cell,
approximately 5 pixel widths from the membrane.
[0193] Image Analysis
[0194] The fluorescent images were processed using MetaMorph
imaging software (Universal Imaging Corporation, Downington Pa.).
Fluorescent accumulation was established by measuring the average
pixel intensity of time resolved fluorescent images within a
specified region; regions of interest are identified by the DIC
image. Average pixel intensity is used to normalize between cells
of different sizes. Parental (HEK-293) cells possess endogenous
mechanisms for ASP.sup.+ accumulation, therefore, NET mediated
ASP.sup.+ accumulation is defined as the fluorescence of HEK-hNET
cells minus the fluorescence of HEK-293cells. All cells are
subtracted against auto-fluorescence.
Example 2
ASP.sup.+ Uptake bv NET, DAT & SERT
[0195] In the present Example the inventors demonstrate that the
fluorescent molecule, ASP.sup.+, is a substrate for NET, DAT and
SERT. The inventors have shown that cells exogenously expressing
NET, SERT and DAT accumulate ASP.sup.+ over parental HEK-293 cells,
indicating the use of ASP.sup.+ as a powerful tool for the
investigation of neurotransmitter transporters, especially the
monoamine transporters. Although this example describes specific
details and measurements with regard to NET, one of skill in the
art will realize that similar analysis can be performed on DAT,
SERT as well as ET. The present invention contemplates quantifying
ASP.sup.+ fluorescence to estimate NET, DAT, SERT and ET turnover
rates, surface expression, and binding constants in transfected
cells, and further investigation of neurons in tissue culture to
study endogenous NET regulation and function.
[0196] Although ASP.sup.+ is structurally disimilar to the
endogenous substrate, it competes for specific NET-mediated NE
transport. Temporal and spacial information on NET activity show
that ASP.sup.+ accumulation kinetics for the slow phase (phase II)
is similar to radiolabeled NE accumulation kinetics. Spatial
patterning and temperature dependence indicate that the rapid phase
I represents ASP.sup.+ binding to NET and the slow phase II
represents transport. Moreover, measuring transport in single
neurons, ordinarily impossible in single mammalian cells, is
readily achieved with ASP.sup.+. ASP.sup.+ accumulation
demonstrated a punctated pattern similar to NET distribution
determined by immunohistochemistry (Schroeter et al., 2000). The
fluorescent microscopy methods, used herein, also permit analysis
of many cells, while retaining information about single cells.
[0197] MPP and ASP.sup.+ Inhibit NE Accumulation
[0198] To test whether ASP.sup.+ interacts with hNET, the inventors
initially exposed HEK-hNET cells to increasing concentrations of
ASP.sup.+ in the presence of constant amounts of radiolabled NE.
MPP was used as a control, as MPP is a known substrate for NET
(Smith and Levi, 1999) and because MPP has a structure similar to
ASP.sup.+ (FIG. 1). Increasing MPP or ASP.sup.+ inhibits
radiolabled NE accumulation. The inhibition constant (K.sub.i)
values for MPP.sup.+ and ASP.sup.+ were 600.+-.67 nM and 780.+-.77
nM (n=5), respectively; thus MPP.sup.+ and ASP.sup.+ potently
interact with HEK-hNET cells in the sub .mu.M range.
[0199] Cells that Express hNET Accumulate ASP.sup.+
[0200] Confocal slices through the monolayer (FIGS. 2A, 2B &
2C, lower panels) show that ASP.sup.+ accumulation in HEK-HNET
divides into two phases: a rapid phase I ensues immediately after
ASP.sup.+ addition and appears localized to the cell surface (S)
followed by a slower phase II localized to the cell interior (I).
Concomitant differential interference contrast (FIGS. 2A, 2B &
2C, upper panels) help specify the confocal images. In the first
three seconds after adding ASP.sup.+ (FIG. 2B), the cell surface is
noticeably bright, in certain locations, while the internal
compartment remains devoid of ASP.sup.+. As the cell interior
becomes brighter and begins to fill in surface brightness remains
constant. These qualitative features are also observed in DAT- and
SERT-transfected cells. However, the response to ASP.sup.+ is more
robust in hNET-transfected cells, as illustrated in FIGS. 3A-3L.
The weakest responder is SERT (FIGS. 3G-3I), but nonetheless
significantly above parental cells (FIGS. 3J-3K). The remainder of
this example describes only data with hNET-transfected cells.
[0201] FIG. 4 shows that acquiring fluorescence data at 0.3 Hz (or
lower), phase II accumulation is arrested immediately after
removing ASP.sup.+. Although the total intensity decreases due to
ASP removal, the normalized data demonstrate sequestered ASP.sup.+
remains constant. Because ASP.sup.+ binds mitochondria (Stachon et
al., 1996), the inventors contemplate that the flux is
unidirectional and that cells retain the substrate after it enters
the cell. Acquiring data at 12 Hz (or higher) results in a decline
in brightness due to photobleaching. The decay time constant is
linearly proportional to the acquisition rate and likely represents
ASP.sup.+ photo-bleaching, also observed in the absence of cells.
Photo-bleaching thus sets a limit on the frequency of image
acquisition, which under present conditions is 50 ms. Accumulation
in the presence of ASP.sup.+ is independent of sampling rate up to
20 Hz, permitting analysis of phase I during the first 3 seconds
following ASP.sup.+ application. HEK-293 cells also demonstrate
phase I and II; however, both the rapid and slow accumulation phase
are far less intense in parental than in transfected cells.
[0202] Phase I Represents ASP.sup.+ Binding; Phase II Represents
ASP.sup.+ Transport
[0203] A line scan across individual cells at different times, as
shown in FIG. 5A. corroborates that phase I is localized to the
cell surface, as suggested in FIGS. 2A-2C. Notice that rapid
accumulation, which peaks at the cell edges (arrows), remains
essentially constant between 3 and 180 sec., whereas brightness at
the cell center (between arrows) gradually increases over this same
period. Furthermore, accumulation at the center occurs at the same
rate as phase II (compare phase II slope in FIG. 4 with center
elevation in FIG. 5A). HEK-293 cells subjected to similar line
scans do not display similarly localized fluorescence (FIG. 5B).
These data indicate that phase I represents a surface interaction
with hNET and phase II represents ASP.sup.+ transport. To test this
interpretation the inventors measured the anisotropy of light from
cell edges and cell centers. Light from the cell surface
demonstrates significant divergence between horizontal (0.degree.)
and vertical (90.degree.) polarization, whereas light from the
center is less divergent (FIG. 5C). From these data the
fluorescence anisotropy can be calculated (see description supra in
section entitled Materials and Methods) across the line scan (FIG.
5D). The fluorescence anisotropy demonstrates that light from the
edge emits from an effectively immobile source (0.30 <r
<0.32) compared with the center (0.21<r<0.24). Solution
ASP.sup.+ shows even lower anisotropy (0.15). These data indicate
two populations of the fluorescent compound: 1) a rapidly
immobilize ASP.sup.+ located near the cell surface, and 2) a slowly
evolving, mobile pool in the interior, which represent the
transported molecules.
[0204] To segregate the bound and transported populations of
ASP.sup.+ molecules, a potent blocker of NE transport, desipramine
(DS), was administered. As seen qualitatively by comparing FIG. 6A
(without DS) and FIG. 6B (with DS), the blocker virtually
eliminates phase I ASP.sup.+. Because mitochondria sequester
intracellular ASP.sup.+ it remains constant inside the cell after
DS inhibition of transport. If DS interrupts transport at any time
during phase II (arrows), the decrease in fluorescence is
approximately equal to the amplitude of phase I (FIG. 6 C). The
amount of ASP.sup.+ that is sequestered after DS block (bottom
slope) increases linearly over time and is parallel to the rate of
phase II transport. The binding was also found to be less sensitive
to temperature than transport (DeOliveara et al., 1989), indicating
that cold temperature affects phase I less than it affects phase
II. This is demonstrated in FIG. 6D. Furthermore, the biphasic
phase I and phase II pattern does not represent transporter
endocytosis as NET-mediated ASP accumulation is unaffected by a
thirty-minute pre-incubation with concanavilin A or 0.45 mM
sucrose. These data demonstrate that the initial rapid phase I
measures ASP.sup.+ binding, and that the slower phase II represents
ASP.sup.+ transport.
[0205] ASP.sup.+ Pharmacology
[0206] To test whether ASP.sup.+ has pharmacological properties
similar to HNET HEK-hNET cells were pre-incubated for ten minutes
with 10 .mu.M desipramine, 10 .mu.M cocaine or 30 .mu.M NE (Galli
et al., 1995). After pre-incubation with inhibitor alone, 2 .mu.M
ASP.sup.+ was added to the inhibitor solution. In FIG. 7A, data are
separated into phase I and II as previously described. Compared
with injected cells, phase I is a considerably smaller in
non-injected cells and insignificant in the presence of cocaine or
DS, or in the presence of competing NE. Cl.sup.- replacement
enhances phase I, whereas Na.sup.+ replacement does not
significantly alter phase I. Likewise, phase II is abridged in
HEK-293 cells. Phase II is also reduced in the presence of
competing NE and is insignificant in the presence of cocaine.
However, whereas Na replacement dramatically reduces phase II to
DS-insensitive levels, Cl is less effective. Gramicidin, which
reduces the chemical gradients for Na and Cl (White, 1977), was
administered at an intermediate concentration of 10 mg/mL given 15
min. before ASP.sup.+. Gramicidin reduces the slope of both phases.
Finally, phase I and phase II saturate at similar ASP.sup.+ bath
concentrations and have similar Michaelis-Menton constants,
although ASP is slightly more potent for phase I (FIGS. 7B &
7C). Phase I: Vmax=10.5.+-.0.94 AFUs/sec, Km=850.+-.186 nM and
n=1.15.+-.0.30; phase II: Vmax=0.3235 0.014 AFUs/sec, Km=480.+-.60
nM and n=1.51.+-.0.35). The above parameters derived from ASP.sup.+
accumulation are comparable to those obtained with the endogenous
substrate, NE, underscoring ASP.sup.+ s utility as a measure of NET
activity.
[0207] ASP.sup.+ Accumulates in SCG Neurons
[0208] The interactions of ASP.sup.+ with NET in primary tissue
culture neurons were also examined. Superior cervical ganglia (SCG)
neurons endogenously express NET (Schroeter et al., 2000);
therefore, dissociated SCG nerve cells were exposed to 2 .mu.M
ASP.sup.+ after the cells were pre-incubated for 10 minutes in the
presence or absence of 10 .mu.M desipramine. SCG neurons also
contain OCT, which likely contributes to total ASP.sup.+
accumulation; however, DS inhibits NET at 10 .mu.M without
affecting OCT activity (Wu et al., 2000). The difference between
ASP.sup.+ accumulation in the absence and presence of desipramine
establishes specific NET-mediated ASP.sup.+ accumulation in SCG
neurons (see FIG. 8A). As seen in FIGS. 8B and 8C, ASP.sup.+
accumulation in the cell body was similar to HNET-HEK cells, but
accumulation in the neurite was punctuate, similar to hNET
immunohistochemical staining (Schroeter et al., 2000). Thus
ASP.sup.+ is amenable to the investigation of NET not only in
transfected cells but also affords spatial resolution of transport
activity in cultured neurons.
Example 3
Automation of Assays
[0209] In the present Example the inventors have shown that the
methods for measuring transport as well as the screening methods
can be automated to achieve high-throughput analysis of data.
Results of this are depicted in FIG. 9, FIG. 10, and FIG. 11.
[0210] Cell Culture
[0211] HEK-293 cells (American Type Culture Collection, Manassas,
Va.) and HEK-293 cells stably transfected with SERT, NET, or DAT
(Qian et al, 1997) were maintained in monolayer culture at
37.degree. C., 5% CO.sub.2 in Dulbecco's modified Eagle's medium
(DMEM) containing 10% fetal bovine serum, 2 mM glutamine, 100
units/ml penicillin, and 100 .mu.g/ml streptomycin. Medium for the
transfected lines was supplemented with G418 (250 .mu.g/ml).
Trypsin, glutamine, penicillin, streptomycin, G418, and
phosphate-free DMEM were purchased from Life Technologies, Inc. or
obtained from the Vanderbilt Media Core. For all transport assays,
cells were plated on poly-D-lysine (0.1 mg/ml)-coated 96-well
plates. HEK cells were plated at 12,500 cells per well in 96 well
plates, HEK-DAT cells were plated at 15,000 cells per well, HEK-NET
cells were plated at 20,000 per well, and HEK-SERT cells were
plated at 20,000 per well in a volume of 200 uL. HEK, HEK-DAT, and
HEK-SERT cells were grown for 48 hours and HEK-NET cells were grown
for 72 hours to a confluency of .about.90%.
[0212] ASP.sup.+ Uptake Assays
[0213] ASP.sup.+ was obtained from Molecular Probes, Inc. and
dissolved in KRH buffer to indicated concentrations. Trypan Blue
was obtained from Sigma as a 0.4% solution and diluted to a final
concentration of 30 uM in KRH buffer.
[0214] To limit the time delay between the addition of ASP.sup.+
and the measurement of accumulation of ASP.sup.+ during the kinetic
assays, plates were divided in half and assayed one half at a time.
At assay, the medium was removed from wells in the first 6 columns
by aspiration, and the cells were incubated with Trypan Blue in KRH
buffer for 20 minutes at 37.degree. C in room air. Then, medium was
removed from the wells in the last 6 columns and replaced with
Trypan Blue in KRH buffer. Inhibitors were added to appropriate
wells and cells were again incubated at 37.degree. C. in room air.
After incubation, ASP.sup.+ was added to the first 6 columns in 20
.mu.L aliquots at indicated concentrations and accumulated
fluorescence measured and recorded over time by the FLEXStation
ultilizing SoftMax Pro Software. Then, ASP.sup.+ was added to the
last 6 columns and fluorescence accumulation measured and recorded.
Specific uptake was determined by subtracting the amount of
accumulated ASP.sup.+ in the presence of 10 .mu.M Paroxetine, 10
.mu.M final of desipramine, or 0.5 .mu.M of GBR 12909 or GBR 12935
for SERT, NET, and DAT, respectively. Measures were done in
quadruplicate and duplicate in the absence or presence of
inhibitors, respectively. Data from wells treated with inhibitor
were averaged and subtracted from data from untreated wells.
Subtracted data for each concentration of ASP.sup.+ was plotted
versus time in Graphpad Prism and fit with a linear equation to
determine the rate of uptake. Rate of uptake for at least 3
experiments were averaged and plotted versus concentration. Data
was fit with a one-site binding equation to determine K.sub.m and
V.sub.max values.
[0215] Endpoint assays were conducted to evaluate the sensitivity
of ASP.sup.+ uptake to transporter antagonists. At assay, the media
was removed by aspiration and replaced with Trypan Blue in KRH
buffer. Inhibitors of SERT, NE and DAT were added at the indicated
concentrations. Cells were incubated at 37.degree. C. for 10
minutes in room air. The assay was initiated by the addition of 3
.mu.M ASP.sup.+. ASP.sup.+ accumulation was measured after 10
minutes. Specific uptake was determined by subtracting the amount
of accumulated ASP.sup.+ in the presence of saturating amounts of
inhibitors such as 10 .mu.M Paroxetine, 10 .mu.M final of
desipramine, or 0.5 .mu.M of GBR 12909 or GBR 12935 for SERT, NET,
and DAT, respectively. Measures were done in quadruplicate, and the
subtracted values were averaged. Uptake was plotted against the
logarithmic values of inhibitor concentration and fitted with a
one-site competition curve to determine IC.sub.50 values. K.sub.i
values were then calculated using the Cheng-Prusoff equation. The
results for the above described assays are set forth in FIG. 9,
FIG. 10A, FIG. 10B, FIG. 10C, FIG. 11A and FIG. 11B.
[0216] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
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